Serotonin reuptake inhibitors

ABSTRACT

In one aspect, the invention relates to compounds of formula I: 
                         
where X, Y, R 1 , R 2 , R 3 , R 4 , R 4 , and n are as defined in the specification, or a pharmaceutically acceptable salt thereof. The compounds of formula I are serotonin reuptake inhibitors. In another aspect, the invention relates to pharmaceutical compositions comprising such compounds; methods of using such compounds; and process and intermediates for preparing such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/391,758, filed on Oct. 11, 2010; the entire disclosure of which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to 3-(sulfonyl-1-phenoxyethyl)pyrrolidineand 3-(sulfonyl-1-phenoxypropyl)pyrrolidine compounds having activity asserotonin (5-HT) reuptake inhibitors and, in one embodiment, having thecharacteristic of being selectively restricted from the central nervoussystem. The invention also relates to pharmaceutical compositionscomprising such compounds, processes, and intermediates for preparingsuch compounds and methods of using such compounds to treat pulmonaryarterial hypertension and other ailments.

2. State of the Art

Progressive increase in pulmonary vascular resistance (PVR) restrictsblood flow and causes pulmonary arterial hypertension (PAH), whichultimately leads to right heart failure and death. PAH includes primarypulmonary hypertension and pulmonary hypertension associated withcollagen vascular diseases, congenital systemic-to-pulmonary shunts,portal hypertension, and HIV infection. The causes of increased PVRinclude vasoconstriction and vascular remodeling (increased VSMCproliferation/migration/fibrosis and narrowing of vascular lumen). Thegoal of therapy is to improve symptoms and exercise capacity, and,ultimately, survival. Current drug therapies include treatment withprostanoids, calcium channel blockers, endothelin receptor antagonists,and PDE-5 inhibitors. Unfortunately, these drugs typically only providea symptomatic benefit. Therefore, there is an unmet need for drugs thatcan impact PAH disease progression.

There is preclinical evidence for a role of both serotonin(5-hydroxytrypamine, 5-HT) and the serotonin transporter (SERT) in PAH.SERT is highly expressed in human lung and 5-HT, via interaction withSERT, stimulates proliferation of human pulmonary vascular smooth musclecells (HPVSM). The proliferative effects of 5-HT are exaggerated inHPVSM from PAH patients. Serotonin selective reuptake inhibitors (SSRIs)have been shown to prevent or reverse PAH in animal models (Zhu et al.(2009) Clinical and Experimental Pharmacology and Physiology 36(8):e1-e5), and the overexpression and deficiency of SERT increases anddecreases susceptibility to hypoxia-induced PAH in mice, respectively(Shah et al. (2009) Chest 136(3):694-700).

While there are numerous SSRIs available on the market, most aredirected to treating diseases such as depression, anxiety, and othermental health conditions, and thus are designed to primarily enter thecentral nervous system (CNS). Unfortunately, this CNS activity is oftenassociated with adverse effects such as nausea, sexual distraction,insomnia, somnolence and anxiety. Treatment of diseases such as PAH donot require CNS activity. Therefore, it is desirable to design atherapeutic agent that has SERT inhibition activity yet is peripherallyselective, thus potentially avoiding or reducing centrally mediated sideeffects. This invention is directed to that need.

SUMMARY OF THE INVENTION

The present invention provides novel compounds that have been found topossess serotonin reuptake inhibition activity. In one embodiment, thecompounds of the invention are peripherally selective such that theyexist predominantly in the periphery as compared to the central nervoussystem. Accordingly, compounds of the invention are expected to beuseful and advantageous as therapeutic agents for those diseases anddisorders that can be treated by inhibition of the serotonin transporterin the absence of CNS activity, such as pulmonary arterial hypertension(PAH) and anti-platelet therapy.

One aspect of the invention relates to a compound of formula I:

where:

X and Y are —CH—, or X is —CH— and Y is —N—, or X is —N— and Y is —CH—;

R¹ is selected from —C₁₋₆alkyl optionally substituted with 1 to 5 groupsselected from halo, —NHC(O)CH₃, and —C(O)NHCH₃; —C₃₋₆cycloalkyl;—C₁₋₂alkylene-OR; —C₁₋₂alkylene-COOR; —C₀₋₃alkylene-phenyl optionallysubstituted with —NHC(O)CH₃ or —C(O)NHCH₃; and —C₀₋₃alkylene-pyridyl;where R is selected from hydrogen and —C₁₋₃alkyl;

R² is selected from hydrogen, —C₁₋₆alkyl, and —O—C₁₋₆alkyl;

R³ is selected from hydrogen, halo, and cyano;

R⁴ is selected from halo; —C₁₋₆alkyl optionally substituted with 1 to 5fluoro atoms; —O—C₁₋₆alkyl optionally substituted with 1 to 5 fluoroatoms; —C₀₋₁alkylene-phenyl; —O—C₀₋₃alkylene-phenyl; —SO₂—C₁₋₆alkyl;—C(O)NH₂; and —NO₂; and

n is 1 or 2; or a pharmaceutically acceptable salt thereof.

Another aspect of the invention relates to compounds of formula I havinga configuration selected from formulas a, a′, b, b′, c, c′, d, and d′;or enriched in a stereoisomeric form having such configuration.

Yet another aspect of the invention relates to pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier and acompound of the invention. Such compositions may optionally containother active agents. Accordingly, in yet another aspect of theinvention, a pharmaceutical composition comprises a compound of theinvention, a second active agent, and a pharmaceutically acceptablecarrier. Another aspect of the invention relates to a combination ofactive agents, comprising a compound of the invention and a secondactive agent. The compound of the invention can be formulated togetheror separately from the additional agent(s). When formulated separately,a pharmaceutically acceptable carrier may be included with theadditional agent(s). Thus, yet another aspect of the invention relatesto a combination of pharmaceutical compositions, the combinationcomprising: a first pharmaceutical composition comprising a compound offormula I or a pharmaceutically acceptable salt thereof and a firstpharmaceutically acceptable carrier; and a second pharmaceuticalcomposition comprising a second active agent and a secondpharmaceutically acceptable carrier. The invention also relates to a kitcontaining such pharmaceutical compositions, for example where the firstand second pharmaceutical compositions are separate pharmaceuticalcompositions.

Compounds of the invention possess serotonin reuptake inhibitionactivity and are therefore expected to be useful as therapeutic agentsfor treating patients suffering from a disease or disorder that istreated by the inhibition of the serotonin transporter primarily in theperiphery as compared to the CNS. Thus, one aspect of the inventionrelates to a method of treating a disease selected from pulmonaryarterial hypertension, gastrointestinal disorders, cancer, rheumatoidarthritis, osteoarthritis, osteoporosis, and diabetes, comprisingadministering to a patient a therapeutically effective amount of acompound of the invention. In one specific aspect, the invention relatesto a method of treating pulmonary arterial hypertension. Another aspectof the invention relates to a method of treating a patient that is inneed of anti-platelet therapy, comprising administering to a patient atherapeutically effective amount of a compound of the invention.

Still another aspect of the invention relates to a method for inhibitingserotonin reuptake in a mammal comprising administering to the mammal, aserotonin transporter-inhibiting amount of a compound of the invention.

Among the compounds of formula I, compounds of particular interest arethose having a serotonin reuptake inhibition pIC₅₀ value ≧5.0,particularly those having a pIC₅₀≧7.0, and even more particularly thosehaving a pIC₅₀≧8.0.

Since compounds of the invention possess serotonin reuptake inhibitoryactivity, they are also useful as research tools. Accordingly, oneaspect of the invention relates to a method of using a compound of theinvention as a research tool, comprising conducting a biological assayusing a compound of the invention. Compounds of the invention can alsobe used to evaluate new chemical compounds. Thus another aspect of theinvention relates to a method of evaluating a test compound in abiological assay, comprising: (a) conducting a biological assay with atest compound to provide a first assay value; (b) conducting thebiological assay with a compound of the invention to provide a secondassay value; wherein step (a) is conducted either before, after orconcurrently with step (b); and (c) comparing the first assay value fromstep (a) with the second assay value from step (b). Exemplary biologicalassays include a serotonin reuptake assay. Still another aspect of theinvention relates to a method of studying a biological system or samplecomprising serotonin transporters, the method comprising: (a) contactingthe biological system or sample with a compound of the invention; and(b) determining the effects caused by the compound on the biologicalsystem or sample.

The invention also relates to processes and intermediates useful forpreparing compounds of the invention. Accordingly, one aspect of theinvention relates to a process for preparing a compound of formula I,the process comprising deprotecting a compound of formula II to providea compound of formula I, or a salt thereof, where n, and R¹, R³, and R⁴are as defined for the compound of formula I, and P is anamino-protecting group. In other aspects, the invention relates to novelintermediates used in such processes. In one aspect of the invention,such novel intermediates have the formula of II, as defined herein.

Yet another aspect of the invention relates to the use of compounds ofthe invention for the manufacture of medicaments, especially for themanufacture of medicaments useful for treating pulmonary arterialhypertension, for anti-platelet therapy, or for inhibiting serotoninreuptake in a mammal. Still another aspect of the invention relates tothe use of compounds of the invention as research tools. Other aspectsand embodiments of the invention are disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION Definitions

When describing the compounds, compositions, methods and processes ofthe invention, the following terms have the following meanings unlessotherwise indicated. Additionally, as used herein, the singular forms“a,” “an” and “the” include the corresponding plural forms unless thecontext of use clearly dictates otherwise. The terms “comprising”,“including,” and “having” are intended to be inclusive and mean thatthere may be additional elements other than the listed elements. Allnumbers expressing quantities of ingredients, properties such asmolecular weight, reaction conditions, and so forth used herein are tobe understood as being modified in all instances by the term “about,”unless otherwise indicated. Accordingly, the numbers set forth hereinare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At least, and not as anattempt to limit the application of the doctrine of equivalents to thescope of the claims, each number should at least be construed in lightof the reported significant digits and by applying ordinary roundingtechniques.

The term “alkyl” means a monovalent saturated hydrocarbon group that maybe linear or branched. Unless otherwise defined, such alkyl groupstypically contain from 1 to 10 carbon atoms and include, for example,—C₁₋₂alkyl, —C₁₋₃alkyl, —C₁₋₄alkyl, —C₁₋₆alkyl, and —C₁₋₈alkyl.Representative alkyl groups include, by way of example, methyl, ethyl,n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl,n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl and the like.

The term “alkylene” means a divalent saturated hydrocarbon group thatmay be linear or branched. Unless otherwise defined, such alkylenegroups typically contain from 0 to 10 carbon atoms and include, forexample, —C₀₋₁alkylene-, —C₀₋₃alkylene-, and —C₁₋₂alkylene-.Representative alkylene groups include, by way of example: methylene;ethylene; propylene, —(CH₂)₃—, and branched —C₃alkylene such as—CH(CH₃)—CH₂—; butylene, —(CH₂)₄—, and branched —C₄alkylene such as—CH(CH₃)—(CH₂)₂— and —CH₂—CH(CH₃)—CH₂—; pentylene, —(CH₂)₅—, andbranched —C₅alkylene such as —CH(CH₃)—(CH₂)₃— and —CH₂—C(CH₃)₂—CH₂—; andthe like. It is understood that when the alkylene term includes zerocarbons such as —C₀₋₁alkylene- or —C₀₋₃alkylene-, such terms areintended to include the absence of carbon atoms, that is, the alkylenegroup is not present except for a covalent bond attaching the groupsseparated by the alkylene term.

When a specific number of carbon atoms are intended for a particularterm used herein, the number of carbon atoms is shown preceding the termas subscript. For example, the term “—C₁₋₆alkyl” means an alkyl grouphaving from 1 to 6 carbon atoms, and the term “—C₁₋₂alkylene-” means analkylene group having from 1 to 2 carbon atoms, where the carbon atomsare in any acceptable configuration.

The term “cycloalkyl” means a monovalent saturated carbocyclichydrocarbon group. Unless otherwise defined, such cycloalkyl groupstypically contain from 3 to 10 carbon atoms and include, for example,—C₃₋₆cycloalkyl. Representative cycloalkyl groups include, by way ofexample, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

The term “halo” means fluoro, chloro, bromo, and iodo.

As used herein, the phrase “having the formula” or “having thestructure” is not intended to be limiting and is used in the same waythat the term “comprising” is commonly used.

The term “pharmaceutically acceptable” refers to a material that is notbiologically or otherwise unacceptable when used in the invention. Forexample, the term “pharmaceutically acceptable carrier” refers to amaterial that can be incorporated into a composition and administered toa patient without causing unacceptable biological effects or interactingin an unacceptable manner with other components of the composition. Suchpharmaceutically acceptable materials typically have met the requiredstandards of toxicological and manufacturing testing, and include thosematerials identified as suitable inactive ingredients by the U.S. Foodand Drug Administration.

The term “pharmaceutically acceptable salt” means a salt prepared from abase or an acid which is acceptable for administration to a patient,such as a mammal (for example, salts having acceptable mammalian safetyfor a given dosage regime). However, it is understood that the saltscovered by the invention are not required to be pharmaceuticallyacceptable salts, such as salts of intermediate compounds that are notintended for administration to a patient. Pharmaceutically acceptablesalts can be derived from pharmaceutically acceptable inorganic ororganic bases and from pharmaceutically acceptable inorganic or organicacids. In addition, when a compound of formula I contains both a basicmoiety, such as an amine, and an acidic moiety such as a carboxylicacid, zwitterions may be formed and are included within the term “salt”as used herein. Salts derived from pharmaceutically acceptable inorganicbases include ammonium, calcium, copper, ferric, ferrous, lithium,magnesium, manganic, manganous, potassium, sodium, and zinc salts, andthe like. Salts derived from pharmaceutically acceptable organic basesinclude salts of primary, secondary and tertiary amines, includingsubstituted amines, cyclic amines, naturally-occurring amines and thelike, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine,hydrabamine, isopropylamine, lysine, methylglucamine, morpholine,piperazine, piperadine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine, tromethamineand the like. Salts derived from pharmaceutically acceptable inorganicacids include salts of boric, carbonic, hydrohalic (hydrobromic,hydrochloric, hydrofluoric or hydroiodic), nitric, phosphoric, sulfamicand sulfuric acids. Salts derived from pharmaceutically acceptableorganic acids include salts of aliphatic hydroxyl acids (e.g., citric,gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids),aliphatic monocarboxylic acids (e.g., acetic, butyric, formic, propionicand trifluoroacetic acids), amino acids (e.g., aspartic and glutamicacids), aromatic carboxylic acids (e.g., benzoic, p-chlorobenzoic,diphenylacetic, gentisic, hippuric, and triphenylacetic acids), aromatichydroxyl acids (e.g., o-hydroxybenzoic, p-hydroxybenzoic,1-hydroxynaphthalene-2-carboxylic and 3-hydroxynaphthalene-2-carboxylicacids), ascorbic, dicarboxylic acids (e.g., fumaric, maleic, oxalic andsuccinic acids), glucoronic, mandelic, mucic, nicotinic, orotic, pamoic,pantothenic, sulfonic acids (e.g., benzenesulfonic, camphosulfonic,edisylic, ethanesulfonic, isethionic, methanesulfonic,naphthalenesulfonic, naphthalene-1,5-disulfonic,naphthalene-2,6-disulfonic and p-toluenesulfonic acids), xinafoic acid,and the like.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need thereof,i.e., the amount of drug needed to obtain the desired therapeuticeffect. For example, a therapeutically effective amount for treatingpulmonary arterial hypertension is an amount of compound needed to, forexample, reduce, suppress, eliminate, or prevent the symptoms ofpulmonary arterial hypertension or to treat the underlying cause ofpulmonary arterial hypertension. On the other hand, the term “effectiveamount” means an amount sufficient to obtain a desired result, which maynot necessarily be a therapeutic result. For example, when studying asystem comprising a serotonin transporter, an “effective amount” may bethe amount needed to inhibit serotonin reuptake.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as pulmonary arterialhypertension) in a patient, such as a mammal (particularly a human),that includes: (a) preventing the disease or medical condition fromoccurring, i.e., prophylactic treatment of a patient; (b) amelioratingthe disease or medical condition, i.e., eliminating or causingregression of the disease or medical condition in a patient; (c)suppressing the disease or medical condition, i.e., slowing or arrestingthe development of the disease or medical condition in a patient; or (d)alleviating the symptoms of the disease or medical condition in apatient. For example, the term “treating pulmonary arterialhypertension” would include preventing pulmonary arterial hypertensionfrom occurring, ameliorating pulmonary arterial hypertension,suppressing pulmonary arterial hypertension, and alleviating thesymptoms of pulmonary arterial hypertension. The term “patient” isintended to include those mammals, such as humans, that are in need oftreatment or disease prevention, that are presently being treated fordisease prevention or treatment of a specific disease or medicalcondition, as well as test subjects in which compounds of the inventionare being evaluated or being used in an assay, for example an animalmodel.

All other terms used herein are intended to have their ordinary meaningas understood by those of ordinary skill in the art to which theypertain.

In one aspect, this invention relates to novel compounds of formula I:

or a pharmaceutically acceptable salt thereof.

As used herein, the term “compound of the invention” includes allcompounds encompassed by formula I such as the species embodied informulas a-h, a′-h′, II, III, and IV and all other subspecies of suchformulas. In addition, when the compound of the invention contain abasic or acidic group (e.g., amino or carboxyl groups), the compound canexist as a free base, free acid, or in various salt forms. All such saltforms are included within the scope of the invention. Accordingly, thoseskilled in the art will recognize that reference to a compound herein,for example, reference to a “compound of the invention” or a “compoundof formula I” includes a compound of formula I as well aspharmaceutically acceptable salts of that compound unless otherwiseindicated. Furthermore, solvates of compounds of formula I are includedwithin the scope of this invention.

The compounds of formula I contain at least two chiral centers andtherefore, these compounds may be prepared and used in variousstereoisomeric forms. Accordingly, the invention also relates to racemicmixtures, pure stereoisomers (e.g., enantiomers and diastereoisomers),stereoisomer-enriched mixtures, and the like unless otherwise indicated.When a chemical structure is depicted herein without anystereochemistry, it is understood that all possible stereoisomers areencompassed by such structure. Thus, for example, the terms “compound offormula I,” “compounds of formula II,” and so forth, are intended toinclude all possible stereoisomers of the compound. Similarly, when aparticular stereoisomer is shown or named herein, it will be understoodby those skilled in the art that minor amounts of other stereoisomersmay be present in the compositions of the invention unless otherwiseindicated, provided that the utility of the composition as a whole isnot eliminated by the presence of such other isomers. Individualenantiomers may be obtained by numerous methods that are well known inthe art, including stereospecific synthesis, chiral chromatography usinga suitable chiral stationary phase or support, or by chemicallyconverting them into diastereoisomers, separating the diastereoisomersby conventional means such as chromatography or recrystallization, thenregenerating the original enantiomers. Additionally, where applicable,all cis/trans or E/Z isomers (geometric isomers), tautomeric forms andtopoisomeric forms of the compounds of the invention are included withinthe scope of the invention unless otherwise specified.

More specifically, compounds of formula I contain at least two chiralcenters indicated by the symbols * and ** in the following formula:

where X, Y, R¹, R², R³, R⁴, and n are as defined for formula I. In onestereoisomer, both carbon atoms identified by the * and ** symbols havethe (R) configuration. This embodiment of the invention is shown informula a (for n=1) and formula a′ (for n=2):

In this embodiment, compounds have the (R,R) configuration at the * and** carbon atoms or are enriched in a stereoisomeric form having the(R,R) configuration at these carbon atoms.

In another stereoisomer, both carbon atoms identified by the * and **symbols have the (S) configuration. This embodiment of the invention isshown in formula b (for n=1) and formula b′ (for n=2):

In this embodiment, compounds have the (S,S) configuration at the * and** carbon atoms or are enriched in a stereoisomeric form having the(S,S) configuration at these carbon atoms.

In yet another stereoisomer, the carbon atom identified by the symbol *has the (S) configuration and the carbon atom identified by the symbol** has the (R) configuration. This embodiment of the invention is shownin formula c (for n=1) and formula c′ (for n=2):

In this embodiment, compounds have the (S,R) configuration at the * and** carbon atoms or are enriched in a stereoisomeric form having the(S,R) configuration at these carbon atoms.

In still another stereoisomer, the carbon atom identified by thesymbol * has the (R) configuration and the carbon atom identified by thesymbol ** has the (S) configuration. This embodiment of the invention isshown in formula d (for n=1) and formula d′ (for n=2):

In this embodiment, compounds have the (R,S) configuration at the * and** carbon atoms or are enriched in a stereoisomeric form having the(R,S) configuration at these carbon atoms.

Compounds of formula a and b are enantiomers and therefore, in separateaspects, this invention relates to each individual enantiomer (i.e., aor b), a racemic mixture of a and b, or an enantiomer-enriched mixtureof a and b comprising predominately a or predominately b. Similarly,compounds of formula c and d are enantiomers and therefore, in separateaspects, this invention relates to each individual enantiomer (i.e., cor d), a racemic mixture of c and d, or an enantiomer-enriched mixtureof c and d comprising predominately c or predominately d.

In some embodiments, in order to optimize the therapeutic activity ofthe compounds of the invention, e.g., to treat pulmonary arterialhypertension, it may be desirable that the carbon atoms identified bythe * and ** symbols have a particular (R,R), (S,S), (S,R), or (R,S)configuration or are enriched in a stereoisomeric form having suchconfiguration. For example, in one embodiment, the compounds of theinvention have the (S,R) configuration of formula c or are enriched in astereoisomeric form having the (S,R) configuration, and in anotherembodiment, the compounds of the invention have the (R,S) configurationof formula d, or are enriched in a stereoisomeric form having the (R,S)configuration. In other embodiments, the compounds of the invention arepresent as racemic mixtures, for example as a mixture of enantiomers offormula a and b, or as a mixture of enantiomers of formula c and d.

The compounds of the invention, as well as those compounds used in theirsynthesis may also include isotopically labeled compounds, i.e., whereone or more atoms have been enriched with atoms having an atomic massdifferent from the atomic mass predominately found in nature. Examplesof isotopes that may be incorporated into the compounds of formula I,for example, include, but are not limited to, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N,¹⁸O, ¹⁷O, ³⁵S, ³⁶Cl, and ¹⁸F. Of particular interest are compounds offormula I enriched in tritium or carbon-14 which can be used, forexample, in tissue distribution studies; compounds of formula I enrichedin deuterium especially at a site of metabolism resulting, for example,in compounds having greater metabolic stability; and compounds offormula I enriched in a positron emitting isotope, such as ¹¹C, ¹⁸F, ¹⁵Oand ¹³N, which can be used, for example, in Positron Emission Topography(PET) studies.

The nomenclature used herein to name the compounds of the invention isillustrated in the Examples herein. This nomenclature has been derivedusing the commercially available AutoNom software (MDL, San Leandro,Calif.).

Representative Embodiments

The following substituents and values are intended to providerepresentative examples of various aspects and embodiments of theinvention. These representative values are intended to further defineand illustrate such aspects and embodiments and are not intended toexclude other embodiments or to limit the scope of the invention. Inthis regard, the representation that a particular value or substituentis preferred is not intended in any way to exclude other values orsubstituents from the invention unless specifically indicated.

In one aspect, this invention relates to compounds of formula I:

X and Y are —CH—, or X is —CH— and Y is —N—, or X is —N— and Y is —CH—.These embodiments can be depicted as formulas Ia, Ib, and Ic,respectively:

In one particular embodiment, X and Y are —CH—.

The “n” integer can be 1 or 2. In one particular embodiment, n is 1.

The R¹ moiety is selected from —C₁₋₆alkyl, —C₃₋₆cycloalkyl,—C₁₋₂alkylene-OR, —C₁₋₂alkylene-COOR, —C₀₋₃alkylene-phenyl, and—C₀₋₃alkylene-pyridyl. The R moiety is selected from hydrogen and—C₁₋₃alkyl. The —C₁₋₆alkyl group in R¹ is optionally substituted with 1to 5 groups selected from halo, —NHC(O)CH₃, and —C(O)NHCH₃. The phenylgroup in R¹ is optionally substituted with —NHC(O)CH₃ or —C(O)NHCH₃.

In one embodiment, R¹ is —C₁₋₆alkyl, examples of which include methyl,ethyl, isopropyl, n-butyl, and t-butyl. In another embodiment, R¹ is—C₁₋₆alkyl substituted with 1 to 5 halo atoms, examples of which include—CF₃, and —CF₂CF₃. In another embodiment, —C₁₋₆alkyl is substituted withone —NHC(O)CH₃ group or one —C(O)NHCH₃ group, examples of which include—(CH₂)₂NHC(O)CH₃ and —(CH₂)₃NHC(O)CH₃. In yet another embodiment, R¹ is—C₃₋₆cycloalkyl, examples of which include cyclopropyl and cyclopentyl.In one embodiment, R¹ is —C₁₋₂alkylene-OR, examples of which include—(CH₂)₂OH(R=H) and —(CH₂)OCH₃ (R=methyl). In one embodiment, R¹ is—C₁₋₂alkylene-COOR, examples of which include —(CH₂)C(O)OCH₃ (R=methyl)and —(CH₂)C(O)OCH₂CH₃ (R=ethyl). In one embodiment, R¹ is—C₀₋₃alkylene-phenyl, i.e., phenyl and benzyl. In another embodiment, R¹is —C₀₋₃alkylene-phenyl where the phenyl is substituted with —NHC(O)CH₃or —C(O)NHCH₃, examples of which include 4-phenylacetamide,3-phenylacetamide, and 2-phenylacetamide, depicted as:

respectively. In one embodiment, R¹ is —C₀₋₃alkylene-pyridyl, examplesof which include 4-pyridyl, 4-pyridyl, and 4-pyridyl, depicted as:

respectively.

In one particular embodiment, R¹ is selected from methyl, ethyl,isopropyl, —(CH₂)₂NHC(O)CH₃, —(CH₂)₂OH, —(CH₂)C(O)OCH₃, phenyl,4-phenylacetamide, and 4-pyridyl. In another particular embodiment, R¹is methyl.

The R² moiety is selected from is selected from hydrogen, —C₁₋₆alkyl,and —O—C₁₋₆alkyl. In one embodiment, R² is hydrogen. In anotherembodiment, R² is —C₁₋₆alkyl, for example —CH₃. In still anotherembodiment, R² is —O—C₁₋₆alkyl, for example —OCH₃.

The R³ moiety is selected from hydrogen, halo, and cyano. In oneembodiment, R³ is hydrogen. In one embodiment, R³ is halo, examples ofwhich include chloro and fluoro. In one embodiment, R³ is cyano. In oneparticular embodiment, R³ is selected from hydrogen and cyano. Inanother particular embodiment, R³ is hydrogen.

The R⁴ moiety is selected from halo, —C₁₋₆alkyl, —C₁₋₆alkyl substitutedwith 1 to 5 fluoro atoms, —O—C₁₋₆alkyl, —O—C₁₋₆alkyl substituted with 1to 5 fluoro atoms, —C₀₋₁alkylene-phenyl, —O—C₀₋₃alkylene-phenyl,—SO₂—C₁₋₆alkyl, —C(O)NH₂, and —NO₂. In one embodiment, R⁴ is halo,examples of which include chloro and fluoro. In one embodiment, R⁴ is—C₁₋₆alkyl, examples of which include methyl, ethyl, isopropyl, n-butyl,and t-butyl. In one embodiment, R⁴ is —C₁₋₆alkyl substituted with 1 to 5fluoro atoms, examples of which include —CH₂F, —CHF₂, —CF₃, and —CF₂CF₃.In one embodiment, R⁴ is —O—C₁₋₆alkyl, examples of which include —OCH₃and —OCH₂CH₃. In one embodiment, R⁴ is —O—C₁₋₆alkyl substituted with 1to 5 fluoro atoms, examples of which include —OCH₂F, —OCHF₂, —OCF₃, and—OCF₂CF₃. In one embodiment, R⁴ is —C₀₋₁alkylene-phenyl, i.e., phenyland benzyl. In one embodiment, R⁴ is —O—C₀₋₃alkylene-phenyl, examples ofwhich include —O-phenyl and —O—CH₂-phenyl. In one embodiment, R⁴ is—SO₂—C₁₋₆alkyl, examples of which include —SO₂—CH₃ and —SO₂—CH₂CH₃. Inone embodiment, R⁴ is —C(O)NH₂. In one embodiment, R⁴ is —NO₂.

In one particular embodiment, R⁴ is selected from halo, —C₁₋₆alkylsubstituted with 1 to 5 fluoro atoms, —O—C₁₋₆alkyl substituted with 1 to5 fluoro atoms, and —NO₂. In another particular embodiment, R⁴ is —CF₃.

In another particular embodiment, R³ and R⁴ combinations are as follows,where R² is hydrogen:

R³ R⁴ H —CF₃ H —OCF₃ H —NO₂ —CN Cl —CN —CF₃

In one particular embodiment, n is 1, and X, Y, R¹, R², R³, and R⁴, areas defined for formula I. This can be depicted as formula III:

Specific embodiments include formulas IIIa, IIIb, and IIIc:

In another particular embodiment, n is 2, and X, Y, R¹, R², R³, and R⁴,are as defined for formula I. This can be depicted as formula IV:

Specific embodiments include formulas IVa, IVb, and IVc:

In one embodiment, R¹ is selected from —C₁₋₆alkyl optionally substitutedwith —NHC(O)CH₃; —C₁₋₂alkylene-OR; —C₁₋₂alkylene-COOR; phenyl optionallysubstituted with —NHC(O)CH₃; and -pyridyl; where R is selected fromhydrogen and —C₁₋₃alkyl; R² is selected from hydrogen, —C₁₋₆alkyl, and—O—C₁₋₆alkyl; R³ is selected from hydrogen and cyano; R⁴ is selectedfrom halo; —C₁₋₆alkyl optionally substituted with 1 to 5 fluoro atoms;—O—C₁₋₆alkyl optionally substituted with 1 to 5 fluoro atoms; and —NO₂;and n is 1 or 2.

In another embodiment, X is —CH— and Y is —N—; R¹ is —C₁₋₆alkyl; R² ishydrogen; R³ is hydrogen; R⁴ is selected from halo and —C₁₋₆alkyloptionally substituted with 1 to 5 fluoro atoms; and n is 1.

In yet another embodiment, X is —N— and Y is —CH—; R² is hydrogen; R¹ is—C₁₋₆alkyl; R³ is hydrogen; R⁴ is selected from halo and —C₁₋₆alkyloptionally substituted with 1 to 5 fluoro atoms; and n is 1.

In addition, particular compounds of formula I that are of interestinclude those set forth in the Examples below, as well as apharmaceutically acceptable salt thereof.

General Synthetic Procedures

Compounds of the invention can be prepared from readily availablestarting materials using the following general methods, the proceduresset forth in the Examples, or by using other methods, reagents, andstarting materials that are known to those skilled in the art. Althoughthe following procedures may illustrate a particular embodiment of theinvention, it is understood that other embodiments of the invention canbe similarly prepared using the same or similar methods or by usingother methods, reagents and starting materials known to those ofordinary skill in the art. It will also be appreciated that wheretypical or preferred process conditions (i.e., reaction temperatures,times, mole ratios of reactants, solvents, pressures, etc.) are given,other process conditions can also be used unless otherwise stated. Whileoptimum reaction conditions will typically vary depending on variousreaction parameters such as the particular reactants, solvents, andquantities used, those of ordinary skill in the art can readilydetermine suitable reaction conditions using routine optimizationprocedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary or desired to preventcertain functional groups from undergoing undesired reactions. Thechoice of a suitable protecting group for a particular functional groupas well as suitable conditions and reagents for protection anddeprotection of such functional groups are well known in the art.Protecting groups other than those illustrated in the proceduresdescribed herein may be used, if desired. For example, numerousprotecting groups, and their introduction and removal, are described inGreene and Wuts, Protecting Groups in Organic Synthesis, Fourth Edition,Wiley, New York, 2006, and references cited therein.

More particularly, in the schemes below, P represents an“amino-protecting group,” a term used herein to mean a protecting groupsuitable for preventing undesired reactions at an amino group.Representative amino-protecting groups include, but are not limited to,t-butoxycarbonyl (BOC), trityl (Tr), benzyloxycarbonyl (Cbz),9-fluorenylmethoxycarbonyl (Fmoc), formyl, benzyl, and the like.Standard deprotection techniques and reagents such as TFA in DCM or HClin 1,4-dioxane, methanol, or ethanol, are used to remove protectinggroups, when present. For example, a BOC group can be removed using anacidic reagent such as hydrochloric acid, trifluoroacetic acid and thelike; while a Cbz group can be removed by employing catalytichydrogenation conditions such as H₂ (1 atm), 10% Pd/C in an alcoholicsolvent.

Suitable inert diluents or solvents for use in these schemes include, byway of illustration and not limitation, tetrahydrofuran (THF),acetonitrile, N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO),toluene, dichloromethane (DCM), chloroform (CHCl₃), and the like.

All reactions are typically conducted at a temperature within the rangeof about −78° C. to 110° C., for example at room temperature. Reactionsmay be monitored by use of thin layer chromatography (TLC), highperformance liquid chromatography (HPLC), and/or LCMS until completion.Reactions may be complete in minutes, may take hours, typically from 1-2hours and up to 48 hours, or days, such as up to 3-4 days. Uponcompletion, the resulting mixture or reaction product may be furthertreated in order to obtain the desired product. For example, theresulting mixture or reaction product may be subjected to one or more ofthe following procedures: dilution (for example with saturated NaHCO₃);extraction (for example, with ethyl acetate, CHCl₃, DCM, aqueous HCl);washing (for example, with DCM, saturated aqueous NaCl, or saturatedaqueous NaHCO₃); drying (for example, over MgSO₄ or Na₂SO₄, or invacuo); filtration; being concentrated (for example, in vacuo); beingredissolved (for example in a 1:1 acetic acid:H₂O solution); and/orpurification (for example by preparative HPLC or reverse phasepreparative HPLC).

By way of illustration, compounds of formula I, as well as their salts,can be prepared by the following schemes, as well as by the proceduresset forth in the examples. In some instances, the * and ** chiralcenters of the protected epoxide and protected alcohol were inferred,based on the hydrolytic kinetic resolution step. Alternately, theassignment of the stereochemistry can be accomplished utilizing theestablished Mosher ester analysis of the intermediate alcohol (see, forexample, Dale and Mosher (1969) J. Org. Chem. 34(9):2543-2549).

While the following schemes may illustrate formation of one particularstereoisomer, the other stereoisomers can be made in a similar manner byusing a starting material having the appropriate stereochemistry.Similarly, while the following schemes may illustrate carrying forward amixture of diastereoisomers, the mixture may be separated so that onlyone stereoisomer is carried forward.

Compounds of formula III (formula I where n is 1) can be prepared asdescribed in Scheme I:

Compounds of formula III can be prepared by reacting the alcohol 1 withthe appropriate aryl or heteroaryl fluoride 2 using a nucleophilicaromatic substitution reaction (S_(N)Ar) to yield compound 3. Thisreaction is typically conducted using sodium hydride (NaH) in a solventsuch as DMF. Compound 3 is then oxidized to yield the protected sulfone4, which is deprotected to yield the desired compound of formula III.Oxidation is typically done with an oxidizing agent such as potassiumperoxomonosulfate in water and methanol.

Compound 1

The aldehyde 6 can be prepared by the2,2,6,6-tetramethyl-1-piperidinyloxy, free radical (TEMPO) mediatedoxidation of the alcohol 5. The alcohol 5, where P is Boc or benzyl, iscommercially available. Alternately, the aldehyde 6 can be prepared byoxidizing the alcohol 5 using any oxidizing agent suitable forconverting a primary alcohol into an aldehyde. Representative oxidizingagents include, for example, dimethyl sulfoxide, Collin's reagent,Corey's reagent, pyridinium dichromate, and the like.

Olefination of the aldehyde 6 by a Wittig reaction yields the alkene 7.Exemplary Wittig reagents include methyltriphenylphosphonium bromide.This step is followed by epoxidation of the alkene using an oxygentransfer catalyst such as methyltrioxorhenium (VII) and hydrogenperoxide as the terminal oxidant to form compound 8.

Compound 8 is then subjected to hydrolytic kinetic resolution to formcompounds 9 and 10, which are then separated to provide the epoxide 9 asa single isomer. The final step involves opening the epoxide 9 using anucleophilic substitution reaction to form compound 1. Examples ofsuitable nucleophiles include NaSR¹ (e.g., sodium methyl mercaptide,where R¹ is methyl). This reaction is typically conducted using asolvent such as THF.

Compound 2

Compound 2 is commercially available or can be prepared by techniquesthat are known in the art. Examples of compound 2 include1-fluoro-4-trifluoromethylbenzene,5-fluoro-2-trifluoromethylbenzonitrile, and 1-fluoro-4-nitrobenzene.

Compounds of formula IV (formula I where n is 2) can be prepared asdescribed in Scheme II:

The first step involves displacement of the tosylate on the tosylateesters 11 and 11′ with compound 12, having the formula R¹—SH, in asuitable solvent such as acetonitrile, to yield alcohols 13 and 13′.Examples of compound 12 include benzenethiol. The alcohols 13 and 13′are then reacted with the appropriate aryl fluoride 2 using anucleophilic aromatic substitution reaction (S_(N)Ar) to yield compounds14 and 14′. Compounds 14 and 14′ are then oxidized to yield theprotected sulfone, which is deprotected to yield the desired compoundsof formula (IV) and (IV′).

Compounds 11 and 11′

The first step in the preparation of compound 6 is the Mukaiyama aldoladdition reaction between the aldehyde 6 and a silyl enol ether to yieldthe esters 15 and 15′ as a mixture of diastereoisomers. This reaction iscatalyzed by a Lewis acid, for example, boron trifluoride, aluminumchloride, or tin tetrachloride. An exemplary silyl enol is1-(t-butyldimethylsilyloxy)-1-methoxyethene, as shown in the scheme.Esters 15 and 15′ are then reduced to form diols 16 and 16′, in asuitable solvent such as tetrahydrofuran. In one embodiment, thereducing agent is lithium tetrahydroaluminate. The next step involvesconverting the hydroxyl group of diols 16 and 16′ into a leaving group.For example, diols 16 and 16′ can undergo tosylation with an appropriatereagent such as p-toluenesulfonyl chloride (TsCl) in a suitable basesuch as triethylenediamine, to form the tosylate esters 11 and 11′. See,for example, Hartung et al. (1997) Synthesis 12:1433-1438. Alternately,compounds 16 and 16′ can be combined with methanesulfonic anhydride inN,N-diisopropylethylamine.

If desired, pharmaceutically acceptable salts of the compounds offormula I can be prepared by contacting the free acid or base form of acompound of formula I with a pharmaceutically acceptable base or acid.

Certain of the intermediates described herein are believed to be noveland accordingly, such compounds are provided as further aspects of theinvention including, for example, compounds of formulas e, e′, f, f, g,g′, h, or h′:

where X, Y, R¹, R², R³, R⁴, and n are as defined for formula I; and Prepresents an amino-protecting group, particularly t-butoxycarbonyl(BOC). In one embodiment of the invention, compounds of the inventioncan be prepared by deprotecting a compound of formula II:

where X, Y, R¹, R², R³, R⁴, and n are as defined for formula I, and Prepresents an amino-protecting group, to provide a compound of formulaI, or a salt thereof. In one particular embodiment, such unprotectedcompounds have the formulas e, e′, f, f, g, g′, h, or h′.

Further details regarding specific reaction conditions and otherprocedures for preparing representative compounds of the invention orintermediates thereof are described in the Examples set forth herein.

Utility

Compounds of the invention possess serotonin reuptake inhibitoryactivity, and in one embodiment, at nanomolar potencies. Thus, thesecompounds have therapeutic utility as serotonin reuptake inhibitors. Inone embodiment, the compounds of the invention act primarily asserotonin reuptake inhibitors, and thus exhibit only minimal orsub-therapeutic activity as norepinephrine reuptake inhibitors and/ordopamine reuptake inhibitors.

The inhibition constant (K_(i)) of a compound is the concentration ofligand in a radioligand binding inhibition assay that would occupy 50%of the transporters if no radioligand were present. K_(i) values can bedetermined from radioligand binding studies with ³H-citalopram asdescribed in Assay 1. These K_(i) values are derived from IC₅₀ values inthe binding assay using the Cheng-Prusoff equation and the K_(d) of theradioligand (Cheng & Prusoff (1973) Biochem. Pharmacol.22(23):3099-3108). Functional IC₅₀ values can be determined in thefunctional inhibition of uptake assays described in Assay 2. These IC₅₀values can be converted to K_(i) values using the Cheng-Prusoff equationand the K_(m) of the transmitter for the transporter. It is notedhowever, that the uptake assay conditions described in Assay 2 are suchthat the IC₅₀ values are very close to the K, values, should amathematical conversion be desired, since the 5-HT concentration used inthe assay is well below its K_(m) for the transporter.

One measure of the affinity of a compound for SERT is the inhibitoryconstant (pK_(i)) for binding to the transporter. The pK_(i) value isthe negative logarithm to base 10 of the K_(i). Compounds of theinvention of particular interest are those having a pK_(i) at SERTgreater than or equal to 5.0, particularly those having a pK_(i) at SERTgreater than or equal to 7.0, and even more particularly those having apK_(i) at SERT greater than or equal to 8.0. Another measure ofserotonin reuptake inhibition is the pIC₅₀ value. In one embodiment,compounds of interest are those having a serotonin reuptake inhibitionpIC₅₀ value greater than or equal to 5.0, particularly those having apIC₅₀ value greater than or equal to 7.0, and even more particularlythose having a pIC₅₀ value greater than or equal to 8.0. Such values canbe determined by techniques that are well known in the art, as well asin the assays described herein.

It is noted that in some cases, compounds of the invention may possessweak serotonin reuptake inhibitory activity. In such cases, those ofordinary skill in the art will recognize that those compounds still haveutility as research tools.

Exemplary assays to determine the serotonin reuptake inhibiting activityof compounds of the invention include by way of illustration and notlimitation, assays that measure SERT binding, for example, as describedin Assay 1 and in Tsuruda et al. (2010) Journal of Pharmacological andToxicological Methods 61(2):192-204. Useful secondary assays includeneurotransmitter uptake assays to measure inhibition of serotonin uptakeinto cells expressing the human or rat recombinant transporter asdescribed in Assay 2, and ex vivo radioligand binding assays that areused to determine the in vivo occupancy of SERT in tissue as describedin Assay 3. Other assays that are useful to evaluate pharmacologicalproperties of test compounds include, but are not limited to, coldligand binding kinetics assays (Motulsky and Mahan (1984) MolecularPharmacol. 25(1):1-9) with membranes prepared from cells expressinghSERT conventional membrane radioligand binding assays usingradiolabeled, for example, tritiated, test compound; radioligand bindingassays using native tissue from, for example rodent or human brain;neurotransmitter uptake assays using human or rodent platelets; andneurotransmitter uptake assays using crude, or pure, synaptosomepreparations from rodent brain.

Exemplary in vivo assays include the rat peripheral serotonin modeldescribed, for example, in Ortiz et al. (1992) British Journal ofPharmacology 105:941-946; and the rat serotonin syndrome modeldescribed, for example, in Izumi et al. (2006) European Journal ofPharmacology 532:258-264. The rat monocrotaline model of pulmonaryarterial hypertension is described, for example, in Kato et al. (2008)J. Cardiovasc. Pharmacol. 51(1):18-23, which is a reliable predictor ofclinical efficacy for the treatment of pulmonary arterial hypertension.Platelet aggregation assays are described for example, in Carneiro etal. (2008) J. Clin. Invest. 118(4):1544-1552. Thrombosis can be measuredby several models, including the arterial thrombosis rodent modeldescribed, in Krekora et al. (1999) Thrombosis Research 96:407-414, andthe rodent model of microarterial anastomosis, described in Nayak et al(2005) Arch Otolaryngol Head Meck Surg. 131:800-803). The mouse hypoxiamodel is also useful to evaluate the compounds of the invention, and isdescribed for example, in Marcos et al., (2003) Am. J. Respir. Crit.Care Med. 168:487-493. The aforementioned assays are useful indetermining the therapeutic utility. Other properties and utilities ofcompounds of the invention can be demonstrated using various in vitroand in vivo assays well known to those skilled in the art.

In one embodiment, the compounds of the invention are peripherallyselective such that they exist predominantly in the periphery (e.g.,plasma) as compared to the central nervous system (e.g., the brain orcerebrospinal fluid), as measured in animal models. One method ofevaluating peripherally selectivity is to determine the ratio of thefree plasma concentration as compared to the free brain or cerebrospinalfluid concentration, measured by the AUC, which is the integral of thedrug concentration after it is administered. In one embodiment,compounds of the invention exhibit a ratio of free plasmaconcentration:free brain concentration greater than 10. One exemplaryassay to measure AUC values is described in Assay 4. Another method ofevaluating peripherally selectivity is to measure the level of SERTinhibition in plasma as compared to the level of SERT inhibition in thebrain. In one embodiment, compounds of the invention exhibit a ratio ofplasma:brain SERT inhibition in the range of about 20:1 to about 80:1(measured in rats). Brain SERT occupancy is another way to measurewhether a compound is peripherally selective. In one embodiment,compounds of the invention exhibit less than 70% brain SERT occupancy(measured in rats).

Compounds of the invention are expected to be useful for the treatmentand/or prevention of medical conditions in which the regulation ofperipheral monoamine transporter function is implicated, in particularthose conditions mediated by or responsive to the inhibition ofserotonin reuptake. Thus, it is expected that patients suffering from adisease or disorder that is treated by the inhibition of the serotonintransporter can be treated by administering a therapeutically effectiveamount of a serotonin reuptake inhibitor of the invention.

The amount of active agent administered per dose or the total amountadministered per day may be predetermined or it may be determined on anindividual patient basis by taking into consideration numerous factors,including the nature and severity of the patient's condition, thecondition being treated, the age, weight, and general health of thepatient, the tolerance of the patient to the active agent, the route ofadministration, pharmacological considerations such as the activity,efficacy, pharmacokinetics and toxicology profiles of the active agentand any secondary agents being administered, and the like. Treatment ofa patient suffering from a disease or medical condition (such aspulmonary arterial hypertension) can begin with a predetermined dosageor a dosage determined by the treating physician, and will continue fora period of time necessary to prevent, ameliorate, suppress, oralleviate the symptoms of the disease or medical condition. Patientsundergoing such treatment will typically be monitored on a routine basisto determine the effectiveness of therapy. Indicators for the diseasesand conditions described herein, are well known to those skilled in theart, and are readily available to the treating physician. Continuousmonitoring by the physician will ensure that the optimal amount ofactive agent will be administered at any given time, as well asfacilitating the determination of the duration of treatment. This is ofparticular value when secondary agents are also being administered, astheir selection, dosage, and duration of therapy may also requireadjustment. In this way, the treatment regimen and dosing schedule canbe adjusted over the course of therapy so that the lowest amount ofactive agent that exhibits the desired effectiveness is administeredand, further, that administration is continued only so long as isnecessary to successfully treat the disease or medical condition.

Pulmonary Arterial Hypertension (PAH)

Compounds having serotonin reuptake inhibiting activity have been shownto prevent or reverse PAH in animal models. See, for example, Zhu et al.(2009) Clinical and Experimental Pharmacology and Physiology 36(8):e1-e5 and Shah et al. (2009) Chest 136(3):694-700. Thus, compounds ofthe invention are expected to find utility in treating PAH, as well aspotentially finding utility in preventing disease progression. Inaddition, this crystalline compound is expected to find utility intreating PAH associated with chronic obstructive pulmonary disease(COPD); see, for example, Chaouat et al. (2009) Chest 136:3. Fortreatment of PAH, the therapeutically effective amount is typically theamount that is sufficient to lower the pulmonary vascular resistance.Other goals of therapy are to improve a patient's exercise capacity andto decrease mortality associated with PAH. For example, in a clinicalsetting, the therapeutically effective amount can be the amount thatimproves a patient's ability to walk comfortably for a period of 6minutes (covering a distance of approximately 20-40 meters). When usedfor treating this disorder, compounds of the invention may beadministered with secondary agents, including by way of illustration andnot limitation, α-adrenergic antagonists, β₁-adrenergic receptorantagonists, β₂-adrenergic receptor agonists, angiotensin-convertingenzyme inhibitors, anticoagulants, calcium channel blockers, diuretics,endothelin receptor antagonists, PDE-5 inhibitors, prostaglandinanalogs, and combinations thereof.

Thrombosis-Induced Cardiovascular Diseases

Serotonin has been found to play a role in platelet activation (See, forexample, Walther et al. (2003) Cell 115:851-862). Thus, since thecompounds of the invention have serotonin reuptake inhibiting activity,they are expected to find utility in anti-platelet therapy, inparticular for treatment of: thrombosis-induced cardiovascular diseasessuch as: atherosclerosis; cerebrovascular diseases such as stroke;congestive heart failure; coronary artery disease such as angina;myocardial infarction (heart attack) and other forms of ischemic heartdisease; metabolic syndrome (Syndrome X); peripheral vascular disease;pulmonary embolism; thrombosis, including peripheral vascularthrombosis; and thrombotic re-occlusion that may occur after surgery.When used for treating such disorders, compounds of the invention may beadministered in combination with one or more other anti-thromboticagents.

Gastrointestinal Disorders

It has been found that abnormalities in serotonin reuptake can alterenteric serotonergic signaling, leading to sensory, motor, and secretorygut dysfunctions. See, for example, Colucci et al. (2008) Trends inMolecular Medicine 14(7): 295-304. Thus, compounds of the invention areexpected to find utility in treating gastrointestinal disorders in themid or lower gastrointestinal tract. These include, for example,irritable bowel syndrome, diarrhea-predominant irritable bowel syndrome,dyspepsia, functional abdominal bloating, functional constipation, andfunctional diarrhea. When used for treating gastrointestinal disorders,compounds of the invention may be administered with secondary agents,including by way of illustration and not limitation, anti-diarrheals,antispasmodic agents (e.g., anticholinergics and smooth musclerelaxants), and combinations thereof.

Cancer

Recent studies have indicated that the serotonin neurotransmittertransporter plays a role in cancer. See, for example, Gil-Ad et al(2008) International Journal of Oncology 33:277-286 and Amit et al.European Neuropsychopharmacology (2009) 19:726-734. Thus, compounds ofthe invention are expected to find utility as anti-proliferative agentsin treating cancer such as colorectal cancer and leukemia, and may beadministered with secondary agents such as anti-neoplastic agents,anti-proliferative agents, cytotoxic agents, tumor growth inhibitors,and combinations thereof.

Rheumatoid Arthritis

Compounds having serotonin reuptake inhibiting activity have been shownto exhibit anti-inflammatory properties (Roumestan et al. RespiratoryResearch (2007) 8:35), more particularly in a rheumatoid arthritisanimal model (Sacre et al. (2010) Arthritis & Rheumatism 62(3):683-693.Thus, compounds of the invention are expected to find utility in thetreatment of rheumatoid arthritis, and may be administered withsecondary agents such as corticosteroids; disease modifyinganti-rheumatic drugs including hydroxychloroquine, leflunomide,methotrexate, sulfasalazine, gold salts such as intramuscular gold,interleukin-1 receptor antagonist therapies such as anakinra, B celldepleting agents such as rituximab, T-cell costimulatory blocking agentssuch as abatacept, tumor necrosis factor inhibitors such as adalimumab,etanercept, and infliximab, and immunomodulatory and cytotoxic agentssuch as azathioprine, cyclophosphamide, and cyclosporine A;non-steroidal anti-inflammatory agents; and combinations thereof.

Osteoarthritis

The serotonin reuptake inhibitor, duloxetine, has been shown to beuseful in reducing pain severity in patients with osteoarthritis pain ofthe knee. Thus, compounds of the invention are also expected to findutility in the treatment of osteoarthritis, and may be administered withsecondary agents such as analgesics (e.g., acetaminophen),corticosteroids, non-steroidal anti-inflammatory agents; andcombinations thereof.

Osteoporosis

Gut-derived serotonin has been proposed to inhibit bone formation.Recent studies have explored whether affecting the biosynthesis ofgut-derived serotonin could treat osteoporosis by increasing boneformation, and concluded that inhibiting such biosynthesis could becomea new treatment for osteoporosis (Yadav et al. Nature Medicine (2010)16:308-312). Thus, compounds of the invention are also expected to findutility in the treatment of osteoporosis.

Diabetes

The selective serotonin reuptake inhibitor, s-citalopram, has been shownto be useful in treating patients with co-morbid major depression anddiabetes mellitus, showing a potential ability to improve glycemiccontrol (Amsterdam et al. (2006) Neuropsychobiology 54:208-214). Studieswith the selective serotonin reuptake inhibitor, fluvoxamine, suggestthat such compounds may find utility in reducing postprandialhyperglycemia (Moore et al. (2005) Am. J. Physiol. Endocrinol. Metab.288:E556-E563). Thus, compounds of the invention are also expected tofind utility in the treatment of diabetes, and may be administered withorally effective antibiotic secondary agents such as: biguanides such asmetformin; glucagon antagonists; α-glucosidase inhibitors such asacarbose and miglitol; dipeptidyl peptidase IV inhibitors (DPP-IVinhibitors) such as alogliptin, denagliptin, linagliptin, saxagliptin,sitagliptin, and vildagliptin; meglitinides such as repaglinide;oxadiazolidinediones; sulfonylureas such as chlorpropamide, glimepiride,glipizide, glyburide, and tolazamide; thiazolidinediones such aspioglitazone and rosiglitazone; and combinations thereof.

Research Tools

Since compounds of the invention possess serotonin reuptake inhibitionactivity, they are also useful as research tools for investigating orstudying biological systems or samples having serotonin transporters.Any suitable biological system or sample having serotonin transportersmay be employed in such studies, which may be conducted either in vitroor in vivo. Representative biological systems or samples suitable forsuch studies include, but are not limited to, cells, cellular extracts,plasma membranes, tissue samples, isolated organs, mammals (such asmice, rats, guinea pigs, rabbits, dogs, pigs, humans, and so forth), andthe like, with mammals being of particular interest. In one particularembodiment of the invention, serotonin reuptake in a mammal is inhibitedby administering a serotonin reuptake-inhibiting amount of a compound ofthe invention. Compounds of the invention can also be used as researchtools by conducting biological assays using such compounds.

When used as a research tool, a biological system or sample comprising aserotonin transporter is typically contacted with a serotoninreuptake-inhibiting amount of a compound of the invention. After thebiological system or sample is exposed to the compound, the effects ofinhibiting serotonin reuptake are determined using conventionalprocedures and equipment. Exposure encompasses contacting cells ortissue with the compound, administering the compound to a mammal, forexample by i.p. or i.v. administration, by the use of an implantablepump such as the Alzet® osmotic pump, and so forth. This determiningstep may comprise measuring a response, i.e., a quantitative analysis ormay comprise an observation, i.e., a qualitative analysis. Measuring aresponse involves, for example, determining the effects of the compoundon the biological system or sample using conventional procedures andequipment, such as a serotonin reuptake assay. The assay results can beused to determine the activity level as well as the amount of compoundnecessary to achieve the desired result, i.e., a serotoninreuptake-inhibiting amount.

Additionally, compounds of the invention can be used as research toolsfor evaluating other chemical compounds, and thus are useful inscreening assays to discover, for example, new compounds havingserotonin reuptake-inhibiting activity. In this manner, a compound ofthe invention is used as a standard in an assay to allow comparison ofthe results obtained with a test compound and with compounds of theinvention to identify those test compounds that have about equal orsuperior reuptake-inhibiting activity, if any. For example, reuptakedata for a test compound or a group of test compounds is compared to thereuptake data for a compound of the invention to identify those testcompounds that have the desired properties, e.g., test compounds havingreuptake-inhibiting activity about equal or superior to a compound ofthe invention, if any. This aspect of the invention includes, asseparate embodiments, both the generation of comparison data (using theappropriate assays) and the analysis of the test data to identify testcompounds of interest. Thus, a test compound can be evaluated in abiological assay, by a method comprising the steps of: (a) conducting abiological assay with a test compound to provide a first assay value;(b) conducting the biological assay with a compound of the invention toprovide a second assay value; wherein step (a) is conducted eitherbefore, after or concurrently with step (b); and (c) comparing the firstassay value from step (a) with the second assay value from step (b).Exemplary biological assays include serotonin reuptake assays.

Pharmaceutical Compositions and Formulations

Compounds of the invention are typically administered to a patient inthe form of a pharmaceutical composition or formulation. Suchpharmaceutical compositions may be administered to the patient by anyacceptable route of administration including, but not limited to, oral,rectal, vaginal, nasal, inhaled, topical (including transdermal) andparenteral modes of administration. Further, the compounds of theinvention may be administered, for example orally, in multiple doses perday (e.g., twice, three times or four times daily), in a single dailydose, in a twice-daily dose, in a single weekly dose, and so forth. Itwill be understood that any form of the compounds of the invention,(i.e., free base, pharmaceutically acceptable salt, solvate, etc.) thatis suitable for the particular mode of administration can be used in thepharmaceutical compositions discussed herein.

Accordingly, in one embodiment, the invention relates to apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound of the invention. The compositions may containother therapeutic and/or formulating agents if desired. When discussingcompositions, the “compound of the invention” may also be referred toherein as the “active agent,” to distinguish it from other components ofthe formulation, such as the carrier. Thus, it is understood that theterm “active agent” includes compounds of formula I as well aspharmaceutically acceptable salts and solvates of that compound.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of a compound of the invention. Thoseskilled in the art will recognize, however, that a pharmaceuticalcomposition may contain more than a therapeutically effective amount,i.e., bulk compositions, or less than a therapeutically effectiveamount, i.e., individual unit doses designed for multiple administrationto achieve a therapeutically effective amount. Typically, thecomposition will contain from about 0.01-95 wt % of active agent,including, from about 0.01-30 wt %, such as from about 0.01-10 wt %,with the actual amount depending upon the formulation itself, the routeof administration, the frequency of dosing, and so forth. In oneembodiment, a composition suitable for an oral dosage form, for example,may contain about 5-70 wt %, or from about 10-60 wt % of active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable composition for a particular mode of administration iswell within the scope of those skilled in the pharmaceutical arts.Additionally, carriers or excipients used in such compositions arecommercially available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; compressed propellant gases, such aschlorofluorocarbons and hydrofluorocarbons; and other non-toxiccompatible substances employed in pharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with a pharmaceuticallyacceptable carrier and one or more optional ingredients. The resultinguniformly blended mixture may then be shaped or loaded into tablets,capsules, pills, canisters, cartridges, dispensers, and the like, usingconventional procedures and equipment.

In one embodiment, the pharmaceutical compositions are suitable for oraladministration. One exemplary dosing regimen would be an oral dosageform administered once or twice daily. Suitable compositions for oraladministration may be in the form of capsules, tablets, pills, lozenges,cachets, dragees, powders, granules; solutions or suspensions in anaqueous or non-aqueous liquid; oil-in-water or water-in-oil liquidemulsions; elixirs or syrups; and the like; each containing apredetermined amount of the active agent.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills, and the like), the composition will typicallycomprise the active agent and one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate. Solid dosageforms may also comprise: fillers or extenders, such as starches,microcrystalline cellulose, lactose, sucrose, glucose, mannitol, and/orsilicic acid; binders, such as carboxymethylcellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, suchas glycerol; disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and/or sodium carbonate; solution retarding agents, such as paraffin;absorption accelerators, such as quaternary ammonium compounds; wettingagents, such as cetyl alcohol and/or glycerol monostearate; absorbents,such as kaolin and/or bentonite clay; lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoring,and perfuming agents, preservatives and antioxidants may also be presentin the pharmaceutical compositions. Exemplary coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate, polyvinyl acetatephthalate, hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate,carboxymethyl ethyl cellulose, hydroxypropyl methylcellulose acetatesuccinate, and the like. Examples of pharmaceutically acceptableantioxidants include: water-soluble antioxidants, such as ascorbic acid,cysteine hydrochloride, sodium bisulfate, sodium metabisulfate, sodiumsulfite and the like; oil-soluble antioxidants, such as ascorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin,propyl gallate, alpha-tocopherol, and the like; and metal-chelatingagents, such as citric acid, ethylenediamine tetraacetic acid, sorbitol,tartaric acid, phosphoric acid, and the like.

Compositions may also be formulated to provide slow or controlledrelease of the active agent using, by way of example, hydroxypropylmethyl cellulose in varying proportions or other polymer matrices,liposomes and/or microspheres. In addition, the pharmaceuticalcompositions of the invention may contain opacifying agents and may beformulated so that they release the active agent only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes. The active agent canalso be in microencapsulated form, if appropriate, with one or more ofthe above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Liquid dosage formstypically comprise the active agent and an inert diluent, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Suspensions maycontain suspending agents such as, for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminium metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

When intended for oral administration, the pharmaceutical compositionsof the invention may be packaged in a unit dosage form. The term “unitdosage form” refers to a physically discrete unit suitable for dosing apatient, i.e., each unit containing a predetermined quantity of theactive agent calculated to produce the desired therapeutic effect eitheralone or in combination with one or more additional units. For example,such unit dosage forms may be capsules, tablets, pills, and the like.

In another embodiment, the compositions of the invention are suitablefor inhaled administration, and will typically be in the form of anaerosol or a powder. Such compositions are generally administered usingwell-known delivery devices, such as a nebulizer, dry powder, ormetered-dose inhaler. Nebulizer devices produce a stream of highvelocity air that causes the composition to spray as a mist that iscarried into a patient's respiratory tract. An exemplary nebulizerformulation comprises the active agent dissolved in a carrier to form asolution, or micronized and combined with a carrier to form a suspensionof micronized particles of respirable size. Dry powder inhalersadminister the active agent as a free-flowing powder that is dispersedin a patient's air-stream during inspiration. An exemplary dry powderformulation comprises the active agent dry-blended with an excipientsuch as lactose, starch, mannitol, dextrose, polylactic acid,polylactide-co-glycolide, and combinations thereof. Metered-doseinhalers discharge a measured amount of the active agent usingcompressed propellant gas. An exemplary metered-dose formulationcomprises a solution or suspension of the active agent in a liquefiedpropellant, such as a chlorofluorocarbon or hydrofluoroalkane. Optionalcomponents of such formulations include co-solvents, such as ethanol orpentane, and surfactants, such as sorbitan trioleate, oleic acid,lecithin, and glycerin. Such compositions are typically prepared byadding chilled or pressurized hydrofluoroalkane to a suitable containercontaining the active agent, ethanol (if present), and the surfactant(if present). To prepare a suspension, the active agent is micronizedand then combined with the propellant. Alternatively, a suspensionformulation can be prepared by spray drying a coating of surfactant onmicronized particles of the active agent. The formulation is then loadedinto an aerosol canister, which forms a portion of the inhaler.

Compounds of the invention can also be administered parenterally (e.g.,by subcutaneous, intravenous, intramuscular, or intraperitonealinjection). For such administration, the active agent is provided in asterile solution, suspension, or emulsion. Exemplary solvents forpreparing such formulations include water, saline, low molecular weightalcohols such as propylene glycol, polyethylene glycol, oils, gelatin,fatty acid esters such as ethyl oleate, and the like. A typicalparenteral formulation is a sterile pH 4-7 aqueous solution of theactive agent. Parenteral formulations may also contain one or moresolubilizers, stabilizers, preservatives, wetting agents, emulsifiers,and dispersing agents. These formulations may be rendered sterile by useof a sterile injectable medium, a sterilizing agent, filtration,irradiation, or heat.

Compounds of the invention can also be administered transdermally usingknown transdermal delivery systems and excipients. For example, thecompound can be admixed with permeation enhancers, such as propyleneglycol, polyethylene glycol monolaurate, azacycloalkan-2-ones, and thelike, and incorporated into a patch or similar delivery system.Additional excipients including gelling agents, emulsifiers and buffers,may be used in such transdermal compositions if desired.

Secondary Agents

The compounds of the invention may be useful as the sole treatment of adisease or may be combined with one or more other therapeutic agents inorder to obtain the desired therapeutic effect. Thus, in one embodiment,pharmaceutical compositions of the invention contain other drugs thatare co-administered with a compound of the invention. For example, thecomposition may further comprise one or more drugs (also referred to as“secondary agents(s)”). Numerous examples of such therapeutic agents arewell known in the art, and examples are described herein. By combining acompound of the invention with a secondary agent, double therapy can beachieved, i.e., serotonin reuptake inhibitory activity and activityassociated with the secondary agent. Accordingly, in yet another aspectof the invention, a pharmaceutical composition comprises a compound ofthe invention, a second active agent, and a pharmaceutically acceptablecarrier. Third, fourth etc. active agents may also be included in thecomposition. In combination therapy, the amount of compound of theinvention that is administered, as well as the amount of secondaryagents, may be less than the amount typically administered inmonotherapy.

A compound of the invention may be either physically mixed with thesecond active agent to form a composition containing both agents; oreach agent may be present in separate and distinct compositions whichare administered to the patient simultaneously or sequentially. Forexample, a compound of the invention can be combined with a secondactive agent using conventional procedures and equipment to form acombination of active agents comprising a compound of the invention anda second active agent. Additionally, the active agents may be combinedwith a pharmaceutically acceptable carrier to form a pharmaceuticalcomposition comprising a compound of the invention, a second activeagent, and a pharmaceutically acceptable carrier. In this embodiment,the components of the composition are typically mixed or blended tocreate a physical mixture. The physical mixture is then administered ina therapeutically effective amount using any of the routes describedherein.

Alternatively, the active agents may remain separate and distinct beforeadministration to the patient. In this embodiment, the agents are notphysically mixed together before administration but are administeredsimultaneously or at separate times as separate compositions. Suchcompositions can be packaged separately or may be packaged together in akit. When administered at separate times, the secondary agent willtypically be administered less than 24 hours after administration of thecompound of the invention, ranging anywhere from concurrent withadministration of the compound of the invention to about 24 hourspost-dose. This is also referred to as sequential administration. Thus,a compound of the invention can be orally administered simultaneously orsequentially with another active agent using two tablets, with onetablet for each active agent, where sequential may mean beingadministered immediately after administration of the compound of theinvention or at some predetermined time later (e.g., one hour later orthree hours later). Alternatively, the combination may be administeredby different routes of administration, i.e., one orally and the other byinhalation.

In one embodiment, the kit comprises a first dosage form comprising acompound of the invention and at least one additional dosage formcomprising one or more of the secondary agents set forth herein, inquantities sufficient to carry out the methods of the invention. Thefirst dosage form and the second (or third, etc,) dosage form togethercomprise a therapeutically effective amount of active agents for thetreatment or prevention of a disease or medical condition in a patient.

Secondary agent(s), when included, are present in a therapeuticallyeffective amount, i.e., are typically administered in an amount thatproduces a therapeutically beneficial effect when co-administered with acompound of the invention. The secondary agent can be in the form of apharmaceutically acceptable salt, solvate, optically pure stereoisomer,and so forth. Thus, secondary agents listed below are intended toinclude all such forms, and are commercially available or can beprepared using conventional procedures and reagents.

In one embodiment, compounds of the invention are administered incombination with an α-adrenergic antagonist, representative examples ofwhich include, but are not limited to, doxazosin, prazosin, tamsulosin,and terazosin.

Compounds of the invention may also be administered in combination witha β₁-adrenergic receptor antagonist (“β₁-blockers”). Representativeβ₁-blockers include, but are not limited to, acebutolol, alprenolol,amosulalol, arotinolol, atenolol, befunolol, betaxolol, bevantolol,bisoprolol, bopindolol, bucindolol, bucumolol, bufetolol, bufuralol,bunitrolol, bupranolol, bubridine, butofilolol, carazolol, carteolol,carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, epanolol,esmolol, indenolol, labetolol, levobunolol, mepindolol, metipranolol,metoprolol such as metoprolol succinate and metoprolol tartrate,moprolol, nadolol, nadoxolol, nebivalol, nipradilol, oxprenolol,penbutolol, perbutolol, pindolol, practolol, pronethalol, propranolol,sotalol, sufinalol, talindol, tertatolol, tilisolol, timolol,toliprolol, xibenolol, and combinations thereof. In one particularembodiment, the β₁-antagonist is selected from atenolol, bisoprolol,metoprolol, propranolol, sotalol, and combinations thereof. Typically,the β₁-blocker will be administered in an amount sufficient to providefrom about 2-900 mg per dose.

In one embodiment, compounds of the invention are administered incombination with a β₂-adrenergic receptor agonist, representativeexamples of which include, but are not limited to, albuterol,bitolterol, fenoterol, formoterol, indacaterol, isoetharine,levalbuterol, metaproterenol, pirbuterol, salbutamol, salmefamol,salmeterol, terbutaline, and the like. Typically, the β₂-adrenoreceptoragonist will be administered in an amount sufficient to provide fromabout 0.05-500 μg per dose.

Compounds of the invention can also be administered in combination withan angiotensin-converting enzyme (ACE) inhibitor. Representative ACEinhibitors include, but are not limited to, accupril, alacepril,benazepril, benazeprilat, captopril, ceranapril, cilazapril, delapril,enalapril, enalaprilat, fosinopril, fosinoprilat, imidapril, lisinopril,moexipril, monopril, moveltopril, pentopril, perindopril, quinapril,quinaprilat, ramipril, ramiprilat, saralasin acetate, spirapril,temocapril, trandolapril, zofenopril, and combinations thereof. In aparticular embodiment, the ACE inhibitor is selected from: benazepril,enalapril, lisinopril, ramipril, and combinations thereof.

In one embodiment, a compound of the invention is administered incombination with an anticoagulant, representative examples of whichinclude, but are not limited to: coumarines such as warfarin; heparin;and direct thrombin inhibitors such as argatroban, bivalirudin,dabigatran, and lepirudin.

In another embodiment, compounds of the invention are administered incombination with an anti-thrombotic agent. Representativeanti-thrombotic agents include, but are not limited to, aspirin,anti-platelet agents, heparin, and combinations thereof. Anti-plateletagents include: adenosine diphosphate receptor inhibitors such asclopidogrel (e.g., clopidogrel bisulfate), prasugrel, and ticlopidine;phosphodiesterase inhibitors such as cilostazol; glycoprotein IIB/IIIAinhibitors, typically administered intravenously, such as abciximab,defibrotide, eptifibatide, and tirofiban; and adenosine reuptakeinhibitors such as dipyridamole. Exemplary combination anti-thromboticagents include aspirin combined with dipyridamole.

In one embodiment, compounds of the invention are administered incombination with a calcium channel blocker. Representative calciumchannel blockers include, but are not limited to, amlodipine, anipamil,aranipine, barnidipine, bencyclane, benidipine, bepridil, clentiazem,cilnidipine, cinnarizine, diltiazem, efonidipine, elgodipine, etafenone,felodipine, fendiline, flunarizine, gallopamil, isradipine, lacidipine,lercanidipine, lidoflazine, lomerizine, manidipine, mibefradil,nicardipine, nifedipine, niguldipine, niludipine, nilvadipine,nimodipine, nisoldipine, nitrendipine, nivaldipine, perhexyline,prenylamine, ryosidine, semotiadil, terodiline, tiapamil, verapamil, andcombinations thereof. In a particular embodiment, the calcium channelblocker is selected from amlodipine, bepridil, diltiazem, felodipine,isradipine, lacidipine, nicardipine, nifedipine, niguldipine,niludipine, nimodipine, nisoldipine, ryosidine, verapamil, andcombinations thereof. Typically, the calcium channel blocker will beadministered in an amount sufficient to provide from about 2-500 mg perdose.

In one embodiment, compounds of the invention are administered incombination with a diuretic. Representative diuretics include, but arenot limited to: carbonic anhydrase inhibitors such as acetazolamide anddichlorphenamide; loop diuretics, which include sulfonamide derivativessuch as acetazolamide, ambuside, azosernide, bumetanide, butazolamide,chloraminophenamide, clofenamide, clopamide, clorexolone, disulfamide,ethoxolamide, furosemide, mefruside, methazolamide, piretanide,torsemide, tripamide, and xipamide, as well as non-sulfonamide diureticssuch as ethacrynic acid and other phenoxyacetic acid compounds such astienilic acid, indacrinone and quincarbate; osmotic diuretics such asmannitol; potassium-sparing diuretics, which include aldosteroneantagonists such as spironolactone, and Na⁺ channel inhibitors such asamiloride and triamterene; thiazide and thiazide-like diuretics such asalthiazide, bendroflumethiazide, benzylhydrochlorothiazide,benzthiazide, buthiazide, chlorthalidone, chlorothiazide,cyclopenthiazide, cyclothiazide, epithiazide, ethiazide, fenquizone,flumethiazide, hydrochlorothiazide, hydroflumethiazide, indapamide,methylclothiazide, meticrane, metolazone, paraflutizide, polythiazide,quinethazone, teclothiazide, and trichloromethiazide; and combinationsthereof. In a particular embodiment, the diuretic is selected fromamiloride, bumetanide, chlorothiazide, chlorthalidone, dichlorphenamide,ethacrynic acid, furosemide, hydrochlorothiazide, hydroflumethiazide,indapamide, methylclothiazide, metolazone, torsemide, triamterene, andcombinations thereof. The diuretic will be administered in an amountsufficient to provide from about 5-50 mg per day, more typically 6-25 mgper day, with common dosages being 6.25 mg, 12.5 mg, or 25 mg per day.

In a particular embodiment, compounds of the invention are administeredin combination with an endothelin receptor antagonist. Representativeendothelin receptor antagonists include, but are not limited to,selective endothelin receptor antagonists (e.g., sitaxentan,ambrisentan, atrasentan, BQ-123), which affect endothelin A receptors,and dual endothelin receptor antagonists (e.g., bosentan, tezosentan),which affect both endothelin A and B receptors.

In another embodiment, compounds of the invention are administered incombination with a muscarinic antagonist (i.e., anticholinergic agent).Representative muscarinic antagonists include, but are not limited to,atropine, atropine sulfate, atropine oxide, methylatropine nitrate,homatropine hydrobromide, hyoscyamine (d, l) hydrobromide, scopolaminehydrobromide, ipratropium bromide, oxitropium bromide, tiotropiumbromide, methantheline, propantheline bromide, anisotropine methylbromide, clidinium bromide, copyrrolate (Robinul), isopropamide iodide,mepenzolate bromide, tridihexethyl chloride (Pathilone), hexocycliummethylsulfate, cyclopentolate hydrochloride, tropicamide,trihexyphenidyl hydrochloride, pirenzepine, telenzepine, AF-DX 116 andmethoctramine and the like.

In still another embodiment, compounds of the invention are administeredin combination with a non-steroidal anti-inflammatory agent (NSAID).Representative non-steroidal anti-inflammatory agents (NSAIDs) include,but are not limited to: acemetacin, acetyl salicylic acid, alclofenac,alminoprofen, amfenac, amiprilose, amoxiprin, anirolac, apazone,aspirin, azapropazone, benorilate, benoxaprofen, bezpiperylon,broperamole, bucloxic acid, carprofen, clidanac, diclofenac, diflunisal,diftalone, enolicam, etodolac, etoricoxib, fenbufen, fenclofenac,fenclozic acid, fenoprofen, fentiazac, feprazone, flufenamic acid,flufenisal, fluprofen, flurbiprofen, furofenac, ibufenac, ibuprofen,indomethacin, indoprofen, isoxepac, isoxicam, ketoprofen, ketorolac,lofemizole, lornoxicam, meclofenamate, meclofenamic acid, mefenamicacid, meloxicam, mesalamine, miroprofen, mofebutazone, nabumetone,naproxen, niflumic acid, nimesulide, nitroflurbiprofen, olsalazine,oxaprozin, oxpinac, oxyphenbutazone, phenylbutazone, piroxicam,pirprofen, pranoprofen, salsalate, sudoxicam, sulfasalazine, sulindac,suprofen, tenoxicam, tiopinac, tiaprofenic acid, tioxaprofen, tolfenamicacid, tolmetin, triflumidate, zidometacin, zomepirac, and combinationsthereof. In a particular embodiment, the NSAID is selected frometodolac, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac,meloxicam, naproxen, oxaprozin, piroxicam, and combinations thereof.

In a particular embodiment, compounds of the invention are administeredin combination with a phosphodiesterase-5 (PDE-5) inhibitor.Representative PDE-5 inhibitors include, but are not limited to,avanafil, lodenafil, mirodenafil, sildenafil (Revatio®), tadalafil(Adcirca®), vardenafil (Levitra®), and udenafil.

In another embodiment, compounds of the invention are administered incombination with a prostaglandin analog (also referred to as prostanoidsor prostacyclin analogs). Representative prostaglandin analogs include,but are not limited to, beraprost sodium, bimatoprost, epoprostenol,iloprost, latanoprost, travoprost, and treprostinil.

The following formulations illustrate representative pharmaceuticalcompositions of the present invention:

Exemplary Hard Gelatin Capsules for Oral Administration

A compound of the invention (50 g), spray-dried lactose (440 g) andmagnesium stearate (10 g) are thoroughly blended. The resultingcomposition is then loaded into hard gelatin capsules (500 mg ofcomposition per capsule).

Alternately, a compound of the invention (20 mg) is thoroughly blendedwith starch (89 mg), microcrystalline cellulose (89 mg) and magnesiumstearate (2 mg). The mixture is then passed through a No. 45 mesh U.S.sieve and loaded into a hard gelatin capsule (200 mg of composition percapsule).

Exemplary Gelatin Capsule Formulation for Oral Administration

A compound of the invention (100 mg) is thoroughly blended withpolyoxyethylene sorbitan monooleate (50 mg) and starch powder (250 mg).The mixture is then loaded into a gelatin capsule (400 mg of compositionper capsule).

Alternately, a compound of the invention (40 mg) is thoroughly blendedwith microcrystalline cellulose (Avicel PH 103; 259.2 mg) and magnesiumstearate (0.8 mg). The mixture is then loaded into a gelatin capsule(Size #1, White, Opaque) (300 mg of composition per capsule).

Exemplary Tablet Formulation for Oral Administration

A compound of the invention (10 mg), starch (45 mg) and microcrystallinecellulose (35 mg) are passed through a No. 20 mesh U.S. sieve and mixedthoroughly. The granules so produced are dried at 50-60° C. and passedthrough a No. 16 mesh U.S. sieve. A solution of polyvinylpyrrolidone (4mg as a 10% solution in sterile water) is mixed with sodiumcarboxymethyl starch (4.5 mg), magnesium stearate (0.5 mg), and talc (1mg), and this mixture is then passed through a No. 16 mesh U.S. sieve.The sodium carboxymethyl starch, magnesium stearate, and talc are thenadded to the granules. After mixing, the mixture is compressed on atablet machine to afford a tablet weighing 100 mg.

Alternately, a compound of the invention (250 mg) is thoroughly blendedwith microcrystalline cellulose (400 mg), silicon dioxide fumed (10 mg),and stearic acid (5 mg). The mixture is then compressed to form tablets(665 mg of composition per tablet).

Alternately, a compound of the invention (400 mg) is thoroughly blendedwith cornstarch (50 mg), croscarmellose sodium (25 mg), lactose (120mg), and magnesium stearate (5 mg). The mixture is then compressed toform a single-scored tablet (600 mg of compositions per tablet).

Exemplary Suspension Formulation for Oral Administration

The following ingredients are mixed to form a suspension containing 100mg of active agent per 10 mL of suspension:

Ingredients Amount Compound of the invention 1.0 g Fumaric acid 0.5 gSodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 gGranulated sugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum ® K(magnesium 1.0 g aluminum silicate) Flavoring 0.035 mL Colorings 0.5 mgDistilled water q.s. to 100 mL

Exemplary Injectable Formulation for Administration by Injection

A compound of the invention (0.2 g) is blended with 0.4 M sodium acetatebuffer solution (2.0 mL). The pH of the resulting solution is adjustedto pH 4 using 0.5 N aqueous hydrochloric acid or 0.5 N aqueous sodiumhydroxide, as necessary, and then sufficient water for injection isadded to provide a total volume of 20 mL. The mixture is then filteredthrough a sterile filter (0.22 micron) to provide a sterile solutionsuitable for administration by injection.

Exemplary Compositions for Administration by Inhalation

A compound of the invention (0.2 mg) is micronized and then blended withlactose (25 mg). This blended mixture is then loaded into a gelatininhalation cartridge. The contents of the cartridge are administeredusing a dry powder inhaler, for example.

Alternately, a micronized compound of the invention (10 g) is dispersedin a solution prepared by dissolving lecithin (0.2 g) in demineralizedwater (200 mL). The resulting suspension is spray dried and thenmicronized to form a micronized composition comprising particles havinga mean diameter less than about 1.5 μm. The micronized composition isthen loaded into metered-dose inhaler cartridges containing pressurized1,1,1,2-tetrafluoroethane in an amount sufficient to provide about 10 μgto about 500 μg of the compound of the invention per dose whenadministered by the inhaler.

Alternately, a compound of the invention (25 mg) is dissolved in citratebuffered (pH 5) isotonic saline (125 mL). The mixture is stirred andsonicated until the compound is dissolved. The pH of the solution ischecked and adjusted, if necessary, to pH 5 by slowly adding aqueous 1Nsodium hydroxide. The solution is administered using a nebulizer devicethat provides about 10 μg to about 500 μg of the compound of theinvention per dose.

EXAMPLES

The following Preparations and Examples are provided to illustratespecific embodiments of the invention. These specific embodiments,however, are not intended to limit the scope of the invention in any wayunless specifically indicated.

The following abbreviations have the following meanings unless otherwiseindicated and any other abbreviations used herein and not defined havetheir standard meaning:

-   -   AcOH acetic acid    -   Boc t-butoxycarbonyl    -   BSA bovine serum albumin    -   CPME cyclopentyl methyl ether    -   DCM dichloromethane (i.e., methylene chloride)    -   (DHQ)₂Pyr hydroquinine 2,5-diphenyl-4,6-pyrimidinediyl diether    -   DMEM Dulbecco's Modified Eagle's Medium    -   DMF N,N-dimethylformamide    -   DMSO dimethylsulfoxide    -   EDTA ethylenediaminetetraacetic acid    -   EtOAc ethyl acetate    -   EtOH ethanol    -   FBS fetal bovine serum    -   HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid    -   hSERT human serotonin transporter    -   5-HT 5-hydroxytryptamine    -   IPAc isopropyl acetate    -   MeOH methanol    -   MeCN acetonitrile    -   MeTHF 2-methyltetrahydrofuran    -   Oxone® potassium peroxomonosulfate    -   PBS phosphate buffered saline    -   TEMPO 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical    -   TFA trifluoroacetic acid    -   THF tetrahydrofuran

Any other abbreviations used herein but not defined have their standard,generally accepted meaning. Unless noted otherwise, all materials, suchas reagents, starting materials and solvents, were purchased fromcommercial suppliers (such as Sigma-Aldrich, Fluka Riedel-de Haën, andthe like) and were used without further purification.

Preparation 1 (R)-3-vinylpyrrolidine-1-carboxylic Acid t-Butyl Ester

A solution of (S)-3-hydroxymethyl-pyrrolidine-1-carboxylic acid t-butylester (25.0 g, 124 mmol, 1.0 eq.) in DCM (200 mL) was cooled withstirring to 0° C. A solution of potassium bromide (1.5 g, 12.4 mmol, 0.1eq.) and sodium bicarbonate (1.5 g, 17.4 mmol, 0.14 eq.) dissolved inwater (100 mL) was added. After 15 minutes of stirring at 0° C., TEMPO(195.3 mg, 1.2 mmol, 0.01 eq.) was added, followed by the slow additionof sodium hypochlorite (77.3 mL, 1.1 eq.) dropwise keeping the internaltemperature in the 6-8° C. range. The mixture was placed in an ice bathuntil the layers separated. The organic layer was separated and theaqueous layer was extracted with DCM (200 mL). The combined organiclayers were washed with saturated aqueous NaCl (200 mL), dried overNa₂SO₄, filtered, and concentrated to yield crude(S)-3-formylpyrrolidine-1-carboxylic acid t-butyl ester (21.5 g).

A slurry of methyltriphenylphosphonium bromide (16.1 g, 45.2 mmol, 3.0eq.) in THF (50 mL) was cooled to −78° C. 1M Sodiumbis(trimethylsilyl)amide in THF (38.0 mL, 2.8 eq.) was added and themixture was stirred for 30 minutes. A solution of(S)-3-formylpyrrolidine-1-carboxylic acid t-butyl ester (3.0 g, 15.0mmol, 1.0 eq.) in THF (10 mL) was slowly added and the mixture wasstirred at −78° C. for 2 hours. The mixture was warmed to roomtemperature over 3 hours and the reaction was quenched with halfsaturated NH₄Cl (50 mL). The organic layer was washed with saturatedaqueous NaCl (50 mL). The organic layer was collected, dried over MgSO₄,filtered, and concentrated. The resulting oil was slurried in hexanes(50 mL) and the precipitate was filtered off. The filtrate wasconcentrated, diluted with hexanes (25 mL), and chilled at −20° C.overnight. The precipitate was filtered off and the filtrate waspurified by column chromatography (0-100% EtOAc in hexanes) to yield(R)-3-vinylpyrrolidine-1-carboxylic acid t-butyl ester as an oil (2.1g).

¹H-NMR (400 MHz, DMSO): δ (ppm)=5.81-5.71 (m, 1H), 5.13-5.07 (m, 1H),5.05-5.01 (m, 1H), 3.56-3.42 (m, 2H), 3.32-3.24 (m, 1H), 3.08-3.0 (m,1H), 2.83-2.71 (m, 1H), 2.04-1.95 (m, 1H), 1.74-1.60 (m, 1H), 1.45 (s,9H).

Preparation 2 (S)-(S)-3-Oxiranylpyrrolidine-1-carboxylic Acid t-ButylEster and (S)-3-((R)-1,2-Dihydroxyethyl)pyrrolidine-1-carboxylic Acidt-Butyl Ester

To (R)-3-vinylpyrrolidine-1-carboxylic acid t-butyl ester (6.1 g, 30.8mmol, 1.0 eq.) was added 3-pyridinecarbonitrile (320 mg, 3.1 mmol, 0.1eq.) and methyltrioxorhenium (VII) (192 mg, 769 mmol, 0.025 eq.). Themixture was stirred until homogeneous. 30% Hydrogen peroxide (33:77,hydrogen peroxide:H₂O, 4.1 mL, 1.3 eq.) was added, and the mixture wasplaced in a ice water bath and stirred for 2 hours. Additionalmethyltrioxorhenium (VII) (50 mg) was added. The organic layer wascollected and washed with a saturated sodium metabisulfite solutionunder an ice bath. The material was then washed with saturated aqueousNaCl. The organic layer was collected, dried over Na₂SO₄, filtered, andconcentrated. The peroxide layer was carefully quenched by adding icefollowed by adding saturated sodium metabisulfite solution dropwise at0° C. The crude product was purified by column chromatography (0-100%EtOAc in hexanes) to give (S)-3-oxiranylpyrrolidine-1-carboxylic acidt-butyl ester as a yellowish oil (4.2 g).

¹H-NMR (400 MHz, DMSO): δ (ppm)=3.38-3.28 (m, 2H), 3.24-3.12 (m, 1H),3.08-2.98 (m, 1H), 2.94-2.88 (m, 1H), 2.72-2.66 (m, 1H), 2.52-2.46 (m,1H), 2.28-2.00 (m, 1H), 1.98-1.84 (m, 1H), 1.76-1.60 (m, 1H), 1.40 (s,9H).

(S,S)-(+)-N,N′-Bis(3,5-di-t-butylsalicylidene)-1,2-cyclohexanediaminocobalt(II) (24.1 mg, 39.8 μmol) was dissolved in toluene (2.0 mL). AcOH wasadded (4.5 μL), and the resulting mixture was stirred at roomtemperature for 1 hour under air. The mixture was then concentrated anddried under vacuum. (S)-3-Oxiranylpyrrolidine-1-carboxylic acid t-butylester (1.7 g, 8.0 mmol) was added, followed by water (71.8 μL), and theresulting mixture was stirred vigorously for 6 hours. Hexanes (10 mL)was added. The hexanes layer was then collected. The bottom layer ofblack oil containing the diol was washed twice with hexanes (10 mL). Thehexane washes were combined and allowed to settle at −20° C. overnight.The hexane solution was then decanted and concentrated.

The resulting oil was purified by flash column chromatography (0-100%EtOAc in hexanes) to yield (S)-(S)-3-oxiranylpyrrolidine-1-carboxylicacid t-butyl ester as an oil (750 μg): ¹H-NMR (400 MHz, DMSO): δ(ppm)=3.54-3.27 (m, 2H), 3.41-3.28 (m, 1H), 3.25-3.11 (m, 1H), 3.11-2.97(m, 1H), 2.91 (ddd, J=6.6, 4.0, 2.7 Hz, 1H), 2.69 (dd, J=4.8, 4.1 Hz,1H), 2.55-2.50 (m, 1H), 2.06 (dq, J=14.1, 7.2 Hz, 1H), 1.98-1.84 (m,1H), 1.78-1.60 (m, 1H), 1.39 (s, 9H).

The black oils were dissolved in DCM and purified by flash columnchromatography (10-15% MeOH in DCM) to yield(S)-3-((R)-1,2-dihydroxyethyl)pyrrolidine-1-carboxylic acid t-butylester as a black oil (0.680 μg): ¹H-NMR (400 MHz, DMSO): δ (ppm)=5.75(s, 1H), 4.63 (d, J=5.1 Hz, 1H), 4.52 (t, J=5.5 Hz, 1H), 3.43-3.23 (m,4H), 3.16-3.00 (m, 1H), 2.94 (dt, J=13.4, 10.1 Hz, 1H), 2.17 (qd,J=15.2, 7.3 Hz, 1H), 1.96-1.74 (m, 1H), 1.75-1.54 (m, 1H), 1.38 (s, 9H).

Preparation 3 (S)-3-((S)-1,2-Dihydroxyethyl)pyrrolidine-1-carboxylicAcid t-Butyl Ester

Potassium ferricyanide(III) (2.3 kg, 6.8 mol, 3.0 eq.) potassiumcarbonate (943.0 g, 6.8 mol, 3.0 eq.), potassium osmate, dihydrate (1.7g, 4.6 mmol), (DHQ)₂Pyr (20 g, 20 mmol, 0.01 eq.), and t-butyl alcohol(6.1 L), were combined in water (6.9 L). The mixture was stirred for 30minutes, then cooled to 3° C. (R)-3-vinylpyrrolidine-1-carboxylic acidt-butyl ester (460 g, 2.3 mol, 1.0 eq.) in t-butyl alcohol (766.7 mL)was added, and the mixture was allowed to cool at 1° C. The mixture wasstirred overnight, and IPC (GC) analysis then indicated that thereaction product was a 2:98 mixture of the SR:SS isomers. The mixturewas warmed to 25° C. and water (3 L) was added, yielding a partialslurry. The layers were allowed to separate. The aqueous layer was backextracted with IPAc (4 L), then stirred for 30 minutes at 25° C. Theaqueous layer was further diluted with water and again back extractedwith IPAc (4 L). The layers were separated and the aqueous was removed.The remaining organic layer was combined with the previously separatedorganic layers, and washed with a saturated aqueous NH₄Cl solution inwater (3 L). The layers were separated and the organic layer wasconcentrated under reduced pressure to afford a thick solution. Thissolution was taken up with IPAc (1.5 L).

The mixture was seeded with(S)-3-((S)-1,2-dihydroxyethyl)pyrrolidine-1-carboxylic acid t-butylester (prepared in a manner as described above) and stirred for 2 hoursat room temperature. Crystallization started after few minutes ofstirring. The slurry was cooled to 2° C. and stirred at that temperaturefor 2 days. The slurry was filtered and the resulting cake was washedwith IPAc (153 mL) then dried under vacuum to yield the title compound(370 g, purity 99%).

The mother liquor (containing a 2:98 mixture of the SR:SS isomers) wasconcentrated to yield a thick oil/solid and was taken up in IPAc (307mL) to form a slurry. This was stirred for 2 hours at room temperature.The slurry was filtered, washed with IPAc (5 mL), and dried to yield anadditional 100 g (purity 97%) of the title compound.

Preparation 4 (S)-(S)-3-Oxiranylpyrrolidine-1-carboxylic Acid t-ButylEster

(S)-3-((S)-1,2-Dihydroxyethyl)pyrrolidine-1-carboxylic acid t-butylester (230 g, 990 mmol, 1.0 eq.) was combined with MeTHF (4.9 kg, 57mol) and cooled to 0° C. 2.0 M Sodium t-butoxide in THF (994 mL, 2.0eq.) was added drop wise over 20 minutes. The mixture was stirred at −1°C. for 15 minutes, then cooled to −7° C. p-Tolylsulfonyl)imidazole (243g, 1.1 mol, 1.1 eq.) was added and the resulting mixture was stirred at0° C. for 2 hours. The reaction was quenched with cold H₂O (5.7 kg, 320mol). Hexanes (1.8 kg, 21 mol) was added and the mixture was warmed to23° C. and stirred for about 30 minutes. The layers were allowed tosettle, the phases were separated, and the reaction vessel was rinsedwith MeTHF (100 mL). The organic layer (˜8 L) was removed and stored at5° C. overnight, then filtered through Na₂SO₄ and concentrated at 60torr to 30 torr with a water bath at 25° C. to yield the title compound(220 g).

Example 1(S)-3-[(S)-2-Methanesulfonyl-1-(4-trifluoromethylphenoxy)ethyl]pyrrolidine

(S)-(S)-3-Oxiranylpyrrolidine-1-carboxylic acid t-butyl ester (150 mg,0.7 mmol, 1.0 eq.) and sodium methyl mercaptide (123 mg, 1.8 mmol, 2.5eq.) were dissolved in THF (0.8 mL). The resulting solution was allowedto react in a microwave reactor for 30 minutes at 100° C. The crudemixture was diluted with hexanes (15 mL) and water (10 mL). The organiclayer was collected, dried under Na₂SO₄, filtered, and concentrated toyield crude compound (a).

Compound (a) was then dissolved in DMF (2.9 mL). NaH (50.6 mg, 2.1 mmol,3.0 eq.) was added and the resulting mixture was stirred for 10 minutes.1-Fluoro-4-trifluoromethylbenzene (268 μL, 3.0 eq.) was added and themixture was heated at 100° C. for 1.5 hours. The mixture was cooled toroom temperature and concentrated under reduced pressure to yield crudecompound (b).

Compound (b) was dissolved in MeOH (42.7 mL) and 0.4 M of Oxone® inwater (17.3 mL). The resultant solution was stirred at room temperaturefor 1 hour then filtered and concentrated under reduced pressure. Thecrude material was dissolved in 1.2M of HCl in EtOH (0.7 mL), stirredovernight at room temperature, and then concentrated under reducedpressure. The crude residue was dissolved in 1:1 AcOH/water (5.0 mL),filtered, and purified by preparative HPLC (10-70% MeCN/H₂O over 50minutes on a 1″ BDS column) to yield the title compound as a TFA salt(26.8 mg). MS m/z: [M+H]⁺ calcd for C₁₄H₁₈F₃NO₃S, 338.10. found 338.0.

¹H-NMR (400 MHz, CDCl₃): δ (ppm)=9.53-9.37 (m, 1H), 9.32 (br. s, 1H),7.60 (d, J=8.8 Hz, 2H), 7.15 (d, J=8.8 Hz, 2H), 5.14 (dd, J=10.5, 5.3Hz, 1H), 3.87-3.63 (m, 2H), 3.62-3.51 (m, 1H), 3.51-3.33 (m, 2H),3.33-3.18 (m, 1H), 3.18-3.00 (m, 1H), 2.95 (s, 3H), 2.33-2.19 (m, 1H),2.03-1.85 (m, 1H).

Monohydrochloride Salt

NaH (120.6 mg, 5.0 mmol, 3.0 eq.) was added to a mixture of compound (a)(438 mg, 1.7 mmol, 1.0 eq.) in DMF (6.9 mL). The resulting mixture wasstirred for 10 minutes. 4-Fluorobenzotrifluoride (638.0 μL, 3.0 eq.) wasadded and the mixture was heated at 100° C. for 1.5 hours. The mixturewas cooled to room temperature and concentrated. The remaining materialwas dissolved in MeOH (101.8 mL) and 0.41 M of Oxone® in water (41.2mL). The resultant solution was stirred at room temperature for 1 hour.The solution was then filtered and concentrated using an evaporator. Thecrude residue was dissolved in 1:1 AcOH/water (15.0 mL), filtered, andpurified by preparative HPLC (10-70% MeCN/H₂O over 50 minutes on a 1″BDS column) in two batches. Both protected and deprotected product wasidentified in the fractions. They were collected separately andlyophilized. The protected fraction was dissolved in 1.2 M of HCl inEtOH (14.0 mL), and the resulting solution was stirred overnight thenconcentrated to yield the title compound as an HCl salt (78 mg). MS m/z:[M+H]⁺338.2.

Preparation of Crystalline Hydrochloride Salt of(S)-3-[(S)-2-Methanesulfonyl-1-(4-trifluoromethylphenoxy)ethyl]pyrrolidineAnhydrous Form I and Anhydrous Form II

(S)-(S)-3-Oxiranylpyrrolidine-1-carboxylic acid t-butyl ester (440.0 g,2.0 mol, 1.0 eq.) was combined with DMF (2 L) at 15° C. While stirring,sodium methyl mercaptide (149 g, 2070 mmol, 0.1 eq.) was added in 5portions over 30 minutes, while maintaining the temperature below 25° C.The mixture was cooled to 18° C., and additional sodium methylmercaptide (7 g, 100 mmol) was added, and the mixture was stirred for 30minutes at 25° C. The mixture was cooled to 0° C. and water (10 kg) wasadded with stirring. IPAc (9 kg, 80 mol) was added, the mixture waswarmed to 20° C. and stirred for 30 minutes to allow for phaseseparation. The aqueous layer was removed and a saturated NH₄Cl solution(0.37:0.63, NH₄Cl:H₂O, 5 L) was added to the organic layer. The mixturewas stirred for 30 minutes at 25° C., the layers were allowed toseparate and the aqueous layer was removed. The organic layer wasconcentrated by rotary evaporation to yield a yellow oil (430 g). Theaqueous layers were maintained at 5° C. for 2 days, warmed to roomtemperature and back extracted with IPAc (1.2 L). The resulting organiclayer was washed with a saturated NH₄Cl solution, the layers wereseparated, and the organic layer was concentrated under reduced pressureto yield a yellow oil (19 g). The two oil residues were taken up in IPAc(1 L) and concentrated under reduced pressure to yield crude containingcompound (a) as a yellow oil (455.0 g) and residual solvents.

DMF (900 g, 10 mol) was added to compound (a) (448.0 g, 1.7 mol, 1.0eq.) and stirred to obtain a homogeneous solution. The solution wasprocessed in two identical batches of equal amount. Half of the mixture(661 g) was combined with 1-fluoro-4-trifluoromethylbenzene (430.0 g,2.6 mol, 1.5 eq.) in DMF (2 L). The mixture was cooled to 3° C. followedby the dropwise addition of 2.0 M of sodium t-butoxide in THF (503.0mL). The mixture was stirred for at least 5 hours, while maintaining thetemperature at 4° C. 2 M of Ammonium chloride in water (10 L) was slowlyadded. The temperature was progressively increased to 25° C. IPAc (7 L)was added and the mixture was stirred for 1 hour, and the phases wereallowed to separate. The aqueous layer was removed, leaving the organiclayer, which was partially concentrated under reduced pressure thenwashed with a NaCl solution to yield crude compound (b). The second halfof the mixture was then processes similarly and the two crudes werecombined to yield crude compound (b).

Crude compound (b) (769.0 g, 1.7 mol, 1.0 eq.) was dissolved intrifluoromethyl benzene (6.5 L) and stirred at 0° C. Ethaneperoxoic acid(1.6 L) was added dropwise, and the resulting mixture was stirred for 1hour as the temperature was raised to room temperature, then stirred foran additional hour at room temperature. The mixture was cooled to 15°C., followed by a slow quench with water (7 L). The phases were allowedto separate and the organic layer was washed with a 7.5% sodiumbicarbonate solution (0.75:9.25, sodium carbonate:H₂O, 7 L). The mixturewas stirred for 30 minutes at 22° C. and the layers were separated. Theorganic layer was dried over Na₂SO₄ and the solvent was removed byrotary evaporation to yield a thick yellow oil. CPME (2.5 L) was addedand mixed at 20° C., followed by addition of heptanes (1.7 L) andpreviously prepared solid compound (c) (1 g). The mixture was stirredfor 1 hour, followed by the slow addition of heptanes (1.6 L). Theresulting thick slurry was filtered and the filter cake was washed withhexanes and dried under nitrogen for 2 days to yield compound (c).

3.0 M HCl in CPME (2.0 L) was slowly added to a mixture of compound (c)(318.9 g, 729.0 mmol, 1.0 eq.) and CPME (1.3 L). The resulting mixturewas stirred at 20° C. overnight. Additional 3.0 M HCl in CPME (2.0 L)was added and the mixture was again stirred at 20° C. overnight. Thereaction vessel was drained and rinsed with CPME (500 mL) and the washeswere combined with the slurry. The slurry was filtered and the cake waswashed with CPME (200 mL), dried under nitrogen overnight, dried undervacuum for 6 hours at 30° C., and at room temperature for 2 days toyield a HCl crystalline material (239.10 g, purity 99%). This materialwas characterized and was designated anhydrous Form I.

A portion of the solution (10 mL) was filtered separately and air driedfor 3 days to yield a HCl crystalline material (1.3 g, purity 99%). Thismaterial was characterized and found to be different from the earlierisolated material, and therefore was designated anhydrous Form II.

The reaction vessel was re-washed with CPME (1.5 L) and H₂O (500 g) tocollect the remaining solids. The solution was concentrated to drynessand taken up in CPME (500 mL), concentrated again, taken up in CPME (500mL) and H₂O (50 g), and allowed to crystallize overnight). The solidswere collected, washed with CPME (50 mL) and dried under nitrogenovernight to yield additional crystalline material (20.1 g, purity 99%).This material was characterized and found to be anhydrous Form II.

Crystalline Hydrochloride Salt of(S)-3-[(S)-2-Methanesulfonyl-1-(4-trifluoromethylphenoxy)ethyl]pyrrolidineMonohydrate

Solid particles of the anhydrous Form I were exposed to 75% RH for oneday. The resulting material was characterized and was found to be amonohydrate.

Example 2

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds2-1 to 2-28, having formula IIIa, were prepared:

(IIIa)

MS m/z: [M+ H]⁺ Ex. R¹ R² R³ R⁴ Formula calcd found 1 —CH₃ H H —CF₃C₁₄H₁₈F₃NO₃S 338.10 338.4 2 —CH₃ H H —CF₃ C₁₄H₁₈F₃NO₃S 338.10 338.3 3—CH₃ H H —CF₃ C₁₄H₁₈F₃NO₃S 338.10 338.3 4 —CH₃ H H —Cl C₁₃H₁₈ClNO₃S304.07 304.0 5 —CH₃ H H —NO₂ C₁₃H₁₈N₂O₅S 315.09 315.0 6 —CH₃ H H —NO₂C₁₃H₁₈N₂O₅S 315.09 315.0 7 —CH₃ H H —NO₂ C₁₃H₁₈N₂O₅S 315.09 315.0 8 —CH₃H H —NO₂ C₁₃H₁₈N₂O₅S 315.09 315.0 9 —CH₃ H H —OCF₃ C₁₄H₁₈F₃NO₄S 354.09354.4 10 —CH₃ H H —OCF₃ C₁₄H₁₈F₃NO₄S 354.09 354.0 11 —CH₃ H H —OCF₃C₁₄H₁₈F₃NO₄S 354.09 354.0 12 —CH₂CH₃ H H —CF₃ C₁₅H₂₀F₃NO₃S 352.11 352.013 —CH(CH₃)₂ H H —CF₃ C₁₆H₂₂F₃NO₃S 366.13 366.0 14 —(CH₂)₂—NHC(O)CH₃ H H—CF₃ C₁₇H₂₃F₃N₂O₄S 409.13 409.0 15 —(CH₂)₂—OH H H —CF₃ C₁₅H₂₀F₃NO₄S368.11 368.0 16 —(CH₂)—C(O)O—CH₃ H H —CF₃ C₁₆H₂₀F₃NO₅S 396.10 396.0 17phenyl H H —CF₃ C₁₉H₂₀F₃NO₃S 400.11 400.0 18 4-phenyl- H H —CF₃C₂₁H₂₃F₃N₂O₄S 457.13 457.0 acetamide 19 4-pyridyl H H —CF₃ C₁₈H₁₉F₃N₂O₃S401.11 401.0 20 —CH₃ H —CN —CF₃ C₁₅H₁₇F₃N₂O₃S 363.09 363.0 21 —CH₃ H —CN—CF₃ C₁₅H₁₇F₃N₂O₃S 363.09 363.0 22 —CH₃ H —CN —Cl C₁₄H₁₇ClN₂O₃S 329.07329.0 23 —CH₃ H —CN —Cl C₁₄H₁₇ClN₂O₃S 329.07 329.0 24 —CH₃ H —CN —ClC₁₄H₁₇ClN₂O₃S 329.07 329.0 25 —CH₃ —OCH₃ H —NO₂ C₁₄H₂₀N₂O₆S 345.10 345.026 —CH₃ —OCH₃ H —NO₂ C₁₄H₂₀N₂O₆S 345.10 345.0 27 —CH₃ —OCH₃ H —NO₂C₁₄H₂₀N₂O₆S 345.10 345.0 28 —CH₃ —CH₃ H —Cl C₁₄H₂₀ClNO₃S 318.09 318.0 1.(S)-3-[(R)-2-Methanesulfonyl-1-(4-trifluoromethylphenoxy)ethyl]pyrrolidine2.(R)-3-[(R)-2-Methanesulfonyl-1-(4-trifluoromethylphenoxy)ethyl]pyrrolidine3.(R)-3-[(S)-2-Methanesulfonyl-1-(4-trifluoromethylphenoxy)ethyl]pyrrolidine4. (S)-3-[(R)-1-(4-Chlorophenoxy)-2-methanesulfonylethyl]pyrrolidine 5.(S)-3-[(S)-2-Methanesulfonyl-1-(4-nitrophenoxy)ethyl]pyrrolidine 6.(S)-3-[(R)-2-Methanesulfonyl-1-(4-nitro-phenoxy)ethyl]pyrrolidine 7.(R)-3-[(S)-2-Methanesulfonyl-1-(4-nitro-phenoxy)ethyl]pyrrolidine 8.(R)-3-[(R)-2-Methanesulfonyl-1-(4-nitro-phenoxy)ethyl]pyrrolidine 9.(S)-3-[(S)-2-Methanesulfonyl-1-(4-trifluoromethoxyphenoxy)ethyl]pyrrolidine10.(S)-3-[(R)-2-Methanesulfonyl-1-(4-trifluoromethoxyphenoxy)ethyl]pyrrolidine11.(R)-3-[(S)-2-Methanesulfonyl-1-(4-trifluoromethoxyphenoxy)ethyl]pyrrolidine12.(S)-3-[(S)-2-Ethanesulfonyl-1-(4-trifluoromethyl-phenoxy)ethyl]pyrrolidine13.(S)-3-[(S)-2-(Propane-2-sulfonyl)-1-(4-trifluoromethylphenoxy)ethyl]pyrrolidine14.N-{2-[(S)-2-(S)-Pyrrolidin-3-yl-2-(4-trifluoromethylphenoxy)ethanesulfonyl]-ethyl}-acetamide15.2-[(S)-2-(S)-Pyrrolidin-3-yl-2-(4-trifluoromethylphenoxy)ethanesulfonyl]ethanol16.[(S)—(S)-Pyrrolidin-3-yl-(4-trifluoromethylphenoxy)ethanesulfonyl]aceticacid methyl ester 17.(S)-3-[(S)-2-Benzenesulfonyl-1-(4-trifluoromethylphenoxy)ethyl]pyrrolidine18.N-{4-[(S)-2-(S)-Pyrrolidin-3-yl-2-(4-trifluoromethylphenoxy)ethanesulfonyl]-phenyl}-acetamide19.4-[(S)-2-(S)-Pyrrolidin-3-yl-2-(4-trifluoromethylphenoxy)ethanesulfonyl]pyridine20.5-((S)-2-Methanesulfonyl-1-(S)-pyrrolidin-3-ylethoxy)-2-trifluoromethyl-benzonitrile21.5-((S)-2-Methanesulfonyl-1-(R)-pyrrolidin-3-ylethoxy)-2-trifluoromethyl-benzonitrile22.2-Chloro-5-((S)-2-methanesulfonyl-1-(S)-pyrrolidin-3-ylethoxy)benzonitrile23.2-Chloro-5-((R)-2-methanesulfonyl-1-(S)-pyrrolidin-3-ylethoxy)benzonitrile24.2-Chloro-5-((S)-2-methanesulfonyl-1-(R)-pyrrolidin-3-ylethoxy)benzonitrile25.(S)-3-[(R)-2-Methanesulfonyl-1-(2-methoxy-4-nitrophenoxy)ethyl]pyrrolidine26.(R)-3-[(S)-2-Methanesulfonyl-1-(2-methoxy-4-nitrophenoxy)ethyl]pyrrolidine27.(R)-3-[(R)-2-Methanesulfonyl-1-(2-methoxy-4-nitrophenoxy)ethyl]pyrrolidine28.(S)-3-[(R)-1-(4-Chloro-2-methylphenoxy)-2-methanesulfonylethyl]pyrrolidine

Preparation 5(R)-3-((S)-1-Hydroxy-2-methylsulfanylethyl)pyrrolidine-1-carboxylic Acidt-Butyl Ester

A solution of (R)-3-hydroxymethyl-pyrrolidine-1-carboxylic acid t-butylester (50.0 g, 248.4 mmol, 1.0 eq.) in DCM (883 mL) was combined withTEMPO (1.9 g, 12.4 mmol, 0.05 eq.) and sodium bromide (2.6 g, 24.8 mmol,0.1 eq.). The solution was cooled to 0° C. A solution of sodiumhypochlorite (272.1 mL, 1.5 eq.) and sodium bicarbonate (31.2 g, 1.5eq.) was added. Upon completion of the reaction, the mixture wasextracted with DCM (3×200 mL) and dried over Na₂SO₄, to yield(R)-3-formylpyrrolidine-1-carboxylic acid t-butyl ester (50 g).

A slurry of methyltriphenylphosphonium bromide (450 g, 1.3 mmol, 3.0eq.) in THF (200 mL) was cooled to −78° C. 2M Sodiumbis(trimethylsilyl)amide (580 mL, 2.8 eq.) was added and the mixture wasstirred for 3 hours. A solution of (R)-3-formylpyrrolidine-1-carboxylicacid t-butyl ester (84 g, 421.6 mmol, 1.0 eq.) in THF (100 mL) was addeddropwise at −65°. Upon completion of the reaction, the product waspurified by column chromatography (200-300 mesh, hexanes:EtOAc=1:3, 3 L)to yield (S)-3-vinyl-pyrrolidine-1-carboxylic acid t-butyl ester (40 g).

To (S)-3-Vinyl-pyrrolidine-1-carboxylic acid t-butyl ester (50 g, 253.4mmol, 1.0 eq.) was added 3-pyridinecarbonitrile (2.7 g, 25.3 mmol, 0.1eq.) and methyltrioxorhenium (VII) (1.3 g, 5.1 mmol, 0.02 eq.). Themixture was stirred until homogeneous. 30% Hydrogen peroxide (11.2 g,329.5 mmol, 1.3 eq.) was added, and the mixture was maintained below 35°C. The mixture was then extracted with EtOAc, washed with water, driedover Na₂SO₄, and purified by column chromatography (hexanes:EtOAc=1:3, 3L) to give (R)-3-Oxiranylpyrrolidine-1-carboxylic acid t-butyl ester (35g).

(R)-3-Oxiranylpyrrolidine-1-carboxylic acid t-butyl ester (30 g, 140.6mmol, 1.0 eq.) was combined with (S,S)-Salen Co(II) (418 mg, 0.7 mmol).Water (⅓ mL) was added and the resulting mixture was stirred vigorouslyfor 6 hours. Hexanes (200 mL) was added. The resulting solids werecrushed and stirred in hexanes to yield a yellow precipitate. Theprecipitate was purified by column chromatography (200-300 mesh,hexanes:EtOAc=1:3, 3 L) to yield(R)-(S)-3-Oxiranylpyrrolidine-1-carboxylic acid t-butyl ester.

(R)-(S)-3-Oxiranylpyrrolidine-1-carboxylic acid t-butyl ester (1.0 g,4.7 mmol, 1.0 eq.) in DMF (10 mL) was combined with sodium methylmercaptide (2.5 g, 14.1 mmol, 3.0 eq.), and the resulting mixture wasstirred at 50° C. for 3 hours under nitrogen. Upon completion of thereaction, the mixture was concentrated under vacuum, extracted withEtOAc, washed with saturated aqueous NaCl and water, dried, andconcentrated. The crude product was purified by preparative HPLC(MeCN:EtOH=4:6) to yield the title compound (700 mg).

Example 32-((S)-2-Methanesulfonyl-1-(R)-pyrrolidin-3-yl-ethoxy)-5-trifluoromethylpyridine

(R)-3-((S)-1-Hydroxy-2-methylsulfanylethyl)pyrrolidine-1-carboxylic acidt-butyl ester (25.0 mg, 95.6 μmol, 1.0 eq.) was dissolved in DMF (1.0mL). NaH (6.9 mg, 287 μmol, 3.0 eq.) was added and the resulting mixturewas stirred for 10 minutes. 2-Fluoro-5-(trifluoromethyl)pyridine (47.4mg, 287 μmol, 3.0 eq.) was added and the mixture was heated at 70° C.for 1.5 hours. The mixture was cooled to room temperature andconcentrated.

The crude material was dissolved in MeOH (5.8 mL) and 0.4 M of Oxone® inwater (2.4 mL). The resultant solution was stirred at room temperaturefor 1 hour then filtered and concentrated under reduced pressure. Thecrude material was dissolved in 1.2M of HCl in EtOH (0.8 mL), stirredovernight at room temperature, and then concentrated. The crude residuewas dissolved in 1:1 AcOH/water (1.5 mL), filtered, and purified bypreparative HPLC (10-70% MeCN/H₂O over 50 minutes on a 1″ BDS column) toyield the title compound as a di-TFA salt (14.2 mg). MS m/z: [M+H]⁺calcd for C₁₃H₁₇F₃N₂O₃S, 339.09. found 339.0.

Example 4

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds4-1 and 4-3, having formula IIIb, were prepared:

(IIIb)

MS m/z: [M+ H]⁺ Ex. R¹ R² R³ R⁴ Formula calcd found 1 —CH₃ H H —ClC₁₂H₁₇ClN₂O₃S 305.07 305.0 2 —CH₃ H H —Cl C₁₂H₁₇ClN₂O₃S 305.07 305.4 3—CH₃ H H —CF₃ C₁₃H₁₇F₃N₂O₃S 339.09 339.0 1.5-Chloro-2-((S)-2-methanesulfonyl-1-(S)-pyrrolidin-3-ylethoxy)pyridine2.5-Chloro-2-((S)-2-methanesulfonyl-1-(R)-pyrrolidin-3-ylethoxy)pyridine3.2-((S)-2-Methanesulfonyl-1-(S)-pyrrolidin-3-yl-ethoxy)-5-trifluoromethyl-pyridine

Example 55-((S)-2-Methanesulfonyl-1-(R)-pyrrolidin-3-yl-ethoxy)-2-trifluoromethylpyridine

(R)-3-((S)-1-Hydroxy-2-methylsulfanylethyl)pyrrolidine-1-carboxylic acidt-butyl ester (25.0 mg, 95.6 μmol, 1.0 eq.) was dissolved in DMF (1.0mL). NaH (6.9 mg, 287 μmol, 3.0 eq.) was added and the resulting mixturewas stirred for 10 minutes. 5-Fluoro-2-(trifluoromethyl)pyridine (47.4mg, 287 μmol, 3.0 eq.) was added and the mixture was heated at 70° C.for 1.5 hours. The mixture was cooled to room temperature andconcentrated.

The crude material was dissolved in MeOH (5.8 mL) and 0.4 M of Oxone® inwater (2.4 mL). The resultant solution was stirred at room temperaturefor 1 hour then filtered and concentrated under reduced pressure. Thecrude material was dissolved in 1.2M of HCl in EtOH (0.8 mL), stirredovernight at room temperature, and then concentrated. The crude residuewas dissolved in 1:1 AcOH/water (1.5 mL), filtered, and purified bypreparative HPLC (10-70% MeCN/H₂O over 50 minutes on a 1″ BDS column) toyield the title compound as a di-TFA salt (16.3 mg). MS m/z: [M+H]⁺calcd for C₁₃H₁₇F₃N₂O₃S, 339.09. found 339.0.

Example 6

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds6-1 to 6-4, having formula IIIc, were prepared:

(IIIc)

MS m/z: [M + H]⁺ Ex. R¹ R² R³ R⁴ Formula calcd found 1 —CH₃ H H —CF₃C₁₃H₁₇F₃N₂O₃S 339.09 339.0 2 —CH₃ H H —CF₃ C₁₃H₁₇F₃N₂O₃S 339.09 339.0 3—CH₃ H H —Cl C₁₂H₁₇ClN₂O₃S 305.07 305.0 4 —CH₃ H H —Cl C₁₂H₁₇ClN₂O₃S305.07 305.0 1.5-((R)-2-Methanesulfonyl-1-(S)-pyrrolidin-3-ylethoxy)-2-trifluoromethylpyridine2.5-((S)-2-Methanesulfonyl-1-(S)-pyrrolidin-3-ylethoxy)-2-trifluoromethylpyridine3.2-Chloro-5-((S)-2-methanesulfonyl-1-(S)-pyrrolidin-3-ylethoxy)pyridine4.2-Chloro-5-((S)-2-methanesulfonyl-1-(R)-pyrrolidin-3-ylethoxy)pyridine

In the compounds described in Preparation 6, and Example 7 and 8, the *chiral center is known and is set forth in the compound name and/ortable. However, the ** chiral center is not known unambiguously.

Preparation 6(S)-3-[(R)-1-Hydroxy-3-(toluene-4-sulfonyloxy)propyl]pyrrolidine-1-carboxylicAcid t-Butyl Ester and(S)-3-[(S)-1-Hydroxy-3-(toluene-4-sulfonyloxy)propyl]pyrrolidine-1-carboxylicAcid t-Butyl Ester

To a solution of (S)-3-hydroxymethyl-pyrrolidine-1-carboxylic acidt-butyl ester (21.6 g, 107 mmol, 1.0 eq.) in DCM (220 mL), was addedTEMPO (300 mg, 2 mmol, 0.02 eq.) and potassium bromide (600 mg, 5 mmol,0.05 eq.) This mixture was cooled at 0° C. and a prechilled (at 0° C.)1:1 mixture of 0.7 M of sodium hypochlorite in water (230 mL, 1.5 eq.)and saturated, aqueous NaHCO₃ (230 ml) was added drop-wise over a periodof 90 minutes. The resultant mixture was extracted with DCM (3×100 mL).The combined organic layers were washed with water (2×100 mL), and thensaturated aqueous NaCl (1×100 mL). The organic layer was dried oversodium sulfate, filtered and concentrated in vacuo to yield(S)-3-formylpyrrolidine-1-carboxylic acid t-butyl ester (14.6 g).

(S)-3-Formylpyrrolidine-1-carboxylic acid t-butyl ester (350 mg, 1.8mmol, 1.0 eq.) was dissolved in DCM (18 mL), and the resulting solutionwas cooled to −78° C. 1-(t-Butyldimethylsilyloxy)-1-methoxyethene (380μL, 1.0 eq.) was added, followed by the addition of boron trifluorideetherate (111 μL, 0.5 eq.) drop-wise via a syringe. The mixture wasstirred at −78° C. for 1 hour then warmed to −40° C. The reaction wasquenched with 1:1 MeOH/triethylamine (1.0 mL). The mixture was warmed toroom temperature and saturated aqueous NaHCO₃ (10 mL) was added. Theaqueous layer was extracted with DCM (3×25 mL). The combined extractswere washed with saturated aqueous NaCl and dried over sodium sulfate.The solvent was evaporated under vacuum and the residue was purified byflash column chromatography on silica gel (0 to 100% EtOAc/hexanes) toyield a 1:1 mixture of the diastereoisomers,(S)-3-((R)-1-hydroxy-2-methoxycarbonylethyl)pyrrolidine-1-carboxylicacid t-butyl ester (145 mg) and(S)-3-((S)-1-hydroxy-2-methoxycarbonylethyl)pyrrolidine-1-carboxylicacid t-butyl ester (145 mg).

For mixture of diastereoisomers: ¹H-NMR (400 MHz, CDCl₃): δ(ppm)=3.95-3.77 (m, 1H), 3.66 (s, 3H), 3.60-3.28 (m, 2H), 3.28-3.03 (m,2H), 3.03-2.86 (m, 1H), 2.59-2.45 (m, 0.5H), 2.45-2.34 (m, 1.5H), 2.18(br. s, 1H), 1.87-1.66 (m, 1H), 1.66-1.48 (m, 1H), 1.40 (s, 9H).

A solution of(S)-3-((R)-1-hydroxy-2-methoxycarbonylethyl)pyrrolidine-1-carboxylicacid t-butyl ester (145 mg, 530 mmol) and(S)-3-((S)-1-hydroxy-2-methoxycarbonylethyl)pyrrolidine-1-carboxylicacid t-butyl ester (145 mg, 530 mmol) in THF (6.2 mL) was added to asolution of 2.0 M of Lithium tetrahydroaluminate in THF (1.6 mL) at 0°C. The mixture was stirred at 0° C. for 1 hour, and the reaction wasquenched with saturated, aqueous NaHCO₃ (5 mL). The mixture was allowedto warm to room temperature. Saturated, aqueous Rochelle's salt (sodiumpotassium tartrate) (20 mL) was added and the mixture was stirred for 1hour. The aqueous layer was extracted with EtOAc (3×15 mL). The organiclayers with saturated aqueous NaCl, dried over Na₂SO₄ and concentratedto yield a mixture of the diastereoisomers,(S)-3-((R)-1,3-dihydroxypropyl)pyrrolidine-1-carboxylic acid t-butylester (135 mg) and(S)-3-((S)-1,3-dihydroxypropyl)pyrrolidine-1-carboxylic acid t-butylester (135 mg).

For mixture of diastereoisomers: ¹H-NMR (400 MHz, CDCl₃): δ(ppm)=4.02-3.90 (m, 1H), 3.90-3.81 (m, 1H), 3.81-3.72 (m, 1H), 3.66-3.35(m, 2H), 3.35-3.05 (m, 2H), 3.05-2.92 (m, 1H), 2.47-2.11 (m, 2H),2.11-2.01 (m, 1H), 1.91-1.81 (m, 0.5H), 1.81-1.51 (m, 2.5H), 1.44 (s,9H).

A mixture of (S)-3-((R)-1,3-dihydroxypropyl)pyrrolidine-1-carboxylicacid t-butyl ester (130 mg, 530 μmol) and(S)-3-((S)-1,3-dihydroxypropyl)pyrrolidine-1-carboxylic acid t-butylester (130 mg, 530 μmol) was dissolved in DCM (4 mL). The mixture wascooled to 0° C. and triethylenediamine (153 mg, 1.4 mmol) was added,followed by p-toluenesulfonyl chloride (219 mg, 1.15 mmol). Theresulting mixture was stirred at 0° C. for 60 minutes. The mixture wasdiluted with EtOAc (10 mL) and washed with water (10 mL). The organiclayer was dried over anhydrous Na₂SO₄, filtered and concentrated byrotary evaporation to yield a mixture of the title diastereoisomers,(S)-3-[(R)-1-hydroxy-3-(toluene-4-sulfonyloxy)propyl]pyrrolidine-1-carboxylicacid t-butyl ester (220 mg) and(S)-3-[(S)-1-hydroxy-3-(toluene-4-sulfonyloxy)propyl]pyrrolidine-1-carboxylicacid t-butyl ester (220 mg).

For mixture of diastereoisomers: ¹H-NMR (400 MHz, CDCl₃): δ (ppm)=7.76(d, J=8.0 Hz, 2H), 7.33 (d, J=7.8 Hz, 2H), 4.34-4.18 (m, 1H), 4.18-4.02(m, 1.5H), 3.96 (t, J=8.2 Hz, 0.5H), 3.63 (s, 1H), 3.46 (dd, J=23.5,13.3 Hz, 2H), 3.14 (dd, J=31.2, 17.6 Hz, 2H), 3.01-2.86 (m, 1H), 2.42(s, 3H), 2.14 (br. s, 1H), 2.04-1.72 (m, 2H), 1.72-1.48 (m, 1H), 1.41(dd, J=2.9, 0.8 Hz, 9H).

Example 7

(S)-3-[3-Benzenesulfonyl-1-(4-trifluoromethylphenoxy)propyl]pyrrolidine

A mixture of(S)-3-[(R)-1-hydroxy-3-(toluene-4-sulfonyloxy)propyl]pyrrolidine-1-carboxylicacid t-butyl ester (48 mg, 0.12 mmol, 1.0 eq.) and(S)-3-[(S)-1-hydroxy-3-(toluene-4-sulfonyloxy)propyl]pyrrolidine-1-carboxylicacid t-butyl ester (48.0 mg, 0.12 mmol, 1.0 eq.) was dissolved in MeCN(1.8 mL) and benzenethiol (61.7 μL, 5.0 eq.) was added. The resultingmixture was stirred overnight at 50° C. The mixture was then cooled toroom temperature and diluted with DCM. The mixture was filtered througha microfilter and concentrated to yield a crude mixture of the thioetheralcohol diastereoisomers,(S)-3-((R)-1-hydroxy-3-phenylsulfanylpropyl)pyrrolidine-1-carboxylicacid t-butyl ester and(S)-3-((S)-1-hydroxy-3-phenylsulfanylpropyl)pyrrolidine-1-carboxylicacid t-butyl ester.

This crude mixture of thioether alcohol diastereoisomers was dissolvedin DMF (2.5 mL), and NaH (17.3 mg, 721 μmol) was added. The mixture wasstirred for 10 minutes, and then 4-fluorobenzotrifluoride (91.5 μL) wasadded. The resulting mixture was heated at 100° C. for 3 hours thencooled to room temperature and concentrated to yield a crude mixture ofthioether phenylether diastereoisomers,(S)-3-[(R)-3-phenylsulfanyl-1-(4-trifluoromethylphenoxy)propyl]pyrrolidine-1-carboxylicacid t-butyl ester and(S)-3-[(S)-3-phenylsulfanyl-1-(4-trifluoromethylphenoxy)propyl]pyrrolidine-1-carboxylicacid t-butyl ester.

This crude mixture of thioether phenylether diastereoisomers wasdissolved in MeOH (5.84 mL) and 0.4 M of Oxone® in water (2.95 mL). Theresultant solution was stirred at room temperature for 1 hour thenfiltered and concentrated under reduced pressure. The crude material wasdissolved in 1.2 M of HCl in EtOH (0.7 mL), stirred overnight at roomtemperature, and then concentrated. The crude residue was dissolved in1:1 AcOH/water (18 mL), filtered, and purified in two batches bypreparative HPLC (10-70% MeCN/H₂O over 50 minutes on a 1″ BDS column) toyield the title compound as a mixture of diastereoisomers (46.0 mg) asTFA salts. MS m/z: [M+H]⁺ calcd for C₂₀H₂₂F₃NO₃S, 3414.13. found 414.6.

Example 8

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds8-1 to 8-5, having formula IV, were prepared:

(IV)

MS m/z: [M + H]⁺ Ex. Stereo-chem. R¹ R² R³ R⁴ Formula calcd found 1R-isomer 1 —CH₃ H H —CF₃ C₁₅H₂₀F₃NO₃S 352.11 352.1 2 R-isomer 2 —CH₃ H H—CF₃ C₁₅H₂₀F₃NO₃S 352.11 352.2 3 S-isomer 1 —CH₃ H H —CF₃ C₁₅H₂₀F₃NO₃S352.11 352.1 4 S-isomer 2 —CH₃ H H —CF₃ C₁₅H₂₀F₃NO₃S 352.11 352.1 5 Smixture of —CH—(CH₃)₂ H H —CF₃ C₁₇H₂₄F₃NO₃S 380.14 380.3 isomers 1 and2 1.(R)-3-[3-Methanesulfonyl-1-(4-trifluoromethylphenoxy)propyl]pyrrolidine2.(R)-3-[3-Methanesulfonyl-1-(4-trifluoromethylphenoxy)propyl]pyrrolidine3.(S)-3-[3-Methanesulfonyl-1-(4-trifluoromethylphenoxy)propyl]pyrrolidine4.(S)-3-[3-Methanesulfonyl-1-(4-trifluoromethylphenoxy)propyl]pyrrolidine5.(S)-3-[3-(Propane-2-sulfonyl)-1-(4-trifluoromethylphenoxy)propyl]pyrrolidine

Assay 1 hSERT Binding Assay

Membrane radioligand binding assays were used to measure inhibition oflabeled ligand (³H-citalopram) binding to membranes prepared from cellsexpressing the human recombinant serotonin transporter (hSERT) in orderto determine the pK_(i) values of test compounds at the transporters.

Membrane Preparation from Cells Expressing hSERT

Recombinant human embryonic kidney (HEK-293) derived cell lines stablytransfected with hSERT, were grown in DMEM medium supplemented with 10%dialyzed FBS, 100 μg/ml penicillin, 100 μg/ml streptomycin, 2 mML-glutamine and 250 μg/ml of the aminoglycoside antibiotic G418, in a 5%CO₂ humidified incubator at 37° C. When cultures reached 80% confluence,the cells were washed thoroughly in PBS (without Ca²⁺ and Mg²⁺) andlifted with 5 mM EDTA in PBS. Cells were pelleted by centrifugation,resuspended in lysis buffer (10 mM Tris-HCl, pH7.5 containing 1 mMEDTA), homogenized, pelleted by centrifugation, and then resuspended in50 mM Tris-HCl, pH 7.5 and 10% sucrose at 4° C. Protein concentration ofthe membrane suspension was determined using a Bio-Rad Bradford ProteinAssay kit. Membranes were snap frozen and stored at −80° C.

Binding Assay

Binding assays were performed in a 96-well assay plate in a total volumeof 200 μl assay buffer (50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, pH 7.4)with 0.5-1 μg membrane protein. Saturation binding studies, to determineradioligand K_(d) values for ³H-citalopram were conducted using 12different radioligand concentrations ranging from 0.005-10 nM.Inhibition assays for determination of pK_(i) values of test compoundswere conducted with ³H-citalopram at 11 different concentrations of testcompound ranging from 10 μM to 100 μM.

Stock solutions (10 mM in DMSO) of test compound were prepared andserial dilutions made using Dilution Buffer (50 mM Tris-HCl, 120 mMNaCl, 5 mM KCl, pH 7.4, 0.1% BSA, 400 μM ascorbic acid). Non-specificradioligand binding was determined in the presence of 1 μM duloxetine(in Dilution Buffer).

Following a 60 minute incubation at 22° C. (or a period sufficient toreach equilibrium), the membranes were harvested by rapid filtrationover a 96-well UniFilter GF/B plate, pretreated with 0.3%polyethyleneimine, and washed 6 times with 300 μl wash buffer (50 mMTris-HCl, 0.9% NaCl). Plates were dried overnight at room temperature,˜45 μl of MicroScint™-20 (Perkin Elmer) added and bound radioactivityquantitated via liquid scintillation spectroscopy Inhibition curves andsaturation isotherms were analyzed using GraphPad Prism Software package(GraphPad Software, Inc., San Diego, Calif.). IC₅₀ values were generatedfrom concentration response curves using the Sigmoidal Dose Response(variable slope) algorithm in Prism GraphPad. K_(d) and B_(max) valuesfor the radioligand were generated from saturation isotherms using theSaturation Binding Global Fit algorithm in Prism GraphPad. pK_(i)(negative decadic logarithm of K_(i)) values for test compounds werecalculated from the best-fit IC₅₀ values, and the K_(d) value of theradioligand, using the Cheng-Prusoff equation (Cheng & Prusoff (1973)Biochem. Pharmacol. 22(23):3099-3108): K_(i)=IC₅₀/(1+[L]/K_(d)), where[L]=concentration radioligand.

Compounds of the invention were tested in this assay and found toexhibit SERT pK_(i) values ≧5.0.

Assay 2 hSERT Neurotransmitter Uptake Assay

Neurotransmitter uptake assays were used to measure inhibition of³H-serotonin (³H-5-HT) uptake into cells expressing hSERT in order todetermine the pIC₅₀ values of test compounds at the transporter, similarto the procedures described in Tsuruda et al. (2010) Journal ofPharmacological and Toxicological Methods 61(2):192-204.

HEK293 cells stably-transfected with human recombinant SERT(HEK293-hSERT) were grown in DMEM medium supplemented with 10% dialyzedfetal bovine serum, 100 μg/ml penicillin, 100 μg/ml streptomycin, 2 mML-glutamine, and 250 μg/ml of the aminoglycoside antibiotic G418. Cellswere incubated in a 5% CO₂, humidified incubator at 37° C.

The neurotransmitter uptake assay was performed in a 96-well assay platein a total volume of 100 μL assay buffer containing HEK293-hSERT cells(7500-10,000 cells/well; plated ˜2 hours prior to assay initiation.Neurotransmitter Transporter Uptake Assay dye (0.5 μM), and elevendifferent concentrations of compound ranging from 10 μM to 100 μM wasused. Non-specific uptake was determined in the presence of indatraline(2.5 μM). The final assay buffer was 12.5 mM Tris-HCl, 5 mM HEPES, 3 mMNaHCO₃, 0.3 mM KH₂PO₄, 0.25 mM Na₂HPO₄, 130 mM NaCl, 5 mM KCl, 1 mMCaCl₂, 0.4 mM MgCl₂, 0.3 mM MgSO₄, 4 mM D-glucose, 0.025% BSA, 0.1 mMascorbic acid, pH 7.4.

In pre-incubation studies, test compound was added to cells for 30minutes at 37° C. prior to the addition of fluorescent substrate. Fordetermination of inhibitory potency, a 10-30 minute endpoint substrateaccumulation was determined by fluorescence spectroscopy using a Safire(Tecan Group Ltd., Männendorf, Switzerland) and analysis was performedas described in Tsuruda et al., supra. For real-time measurements,compounds were either preincubated (as above) or mixed with substrateprior to addition, and kinetic measurements, relative fluorescence units(RFU integrated over 0.5 ms), were made using a cycle time of 1 min.

Compounds of the invention that were tested in this assay were found tohave serotonin reuptake inhibition pIC₅₀ values as follows:

Ex. SERT pIC₅₀ 1 ≧8.0 2-1 ≧8.0 2-2 ≧8.0 2-3 ≧8.0 2-4 ≧7.0 2-5 ≧7.0 2-6≧7.0 2-7 ≧7.0 2-8 ≧7.0 2-9 ≧7.0 2-10 ≧7.0 2-11 ≧8.0 2-12 ≧8.0 2-13 ≧8.02-14 ≧8.0 2-15 ≧8.0 2-16 ≧8.0 2-17 ≧8.0 2-18 ≧8.0 2-19 ≧7.0 2-20 ≧7.02-21 ≧8.0 2-22 ≧8.0 2-23 ≧7.0 2-24 ≧8.0 2-25 ≧7.0 2-26 ≧7.0 2-27 ≧7.02-28 ≧8.0 3 ≧7.0 4-1 ≧7.0 4-2 ≧7.0 4-3 ≧8.0 5 ≧7.0 6-1 ≧7.0 6-2 ≧7.0 6-3≧7.0 6-4 ≧7.0 7 ≧8.0 8-1 ≧7.0 8-2 ≧7.0 8-3 ≧7.0 8-4 ≧7.0 8-5 ≧8.0

Assay 3 Ex Vivo SERT Radioligand Binding Assay

Ex vivo radioligand binding assays are used to determine the in vivooccupancy of SERT, in selected brain regions, following in vivoadministration (acute or chronic) of test compounds. Followingadministration of test compound (by intravenous, intraperitoneal, oral,subcutaneous or other route) at the appropriate dose (0.0001 to 100mg/kg), rats (≧n=4 per group) are euthanized at specific time points (10minutes to 48 hours) by decapitation and the brain dissected on ice.Relevant brain regions are dissected, frozen, and stored at −80° C.until use.

For the ex vivo radioligand binding assay, the initial rate ofassociation of the SERT selective radioligand (³H-citalopram) with ratbrain crude homogenates, prepared from vehicle and test compound-treatedanimals, is monitored (see Hess et al. (2004) J. Pharmacol. Exp. Ther.310(2):488-497). Crude brain tissue homogenates are prepared byhomogenizing frozen tissue pieces in 0.15 ml (per mg wet weight) of 50mM Tris-HCl, 120 mM NaCl, 5 mM KCl, pH 7.4 buffer. Radioligandassociation assays are performed in a 96-well assay plate in a totalvolume of 200 μl assay buffer (50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl,0.025% BSA, pH 7.4) with 650 mg wet weight tissue (equivalent to 25 mgprotein). Homogenates are incubated for up to 5 minutes with³H-citalopram (3 nM) prior to termination of the assay by rapidfiltration over a 96-well UniFilter GF/B plate, pretreated with 0.3%polyethyleneimine. Filters are then washed 6 times with 300 μl washbuffer (50 mM Tris-HCl, 0.9% NaCl, pH 7.4 at 4° C.). Non-specificradioligand binding is determined in the presence of 1 μM duloxetine.The plates are dried overnight at room temperature, ˜45 μl ofMicroScint™-20 (Perkin Elmer) added and bound radioactivity quantitatedvia liquid scintillation spectroscopy. The initial rate of associationof ³H-citalopram is determined by linear regression using GraphPad PrismSoftware package (GraphPad Software, Inc., San Diego, Calif.). Theaverage rate of radioligand association to brain tissue homogenates fromvehicle-treated animals is determined. The % occupancy of test compoundsis then determined using the following equation:% occupancy=100×(1−(initial rate association for test compound-treatedtissue/mean rate association for vehicle-treated tissue))ED₅₀ values are determined by plotting the log 10 of the dose of thetest compound against the % occupancy. ED₅₀ values are generated fromconcentration response curves using the Sigmoidal Dose Response(variable slope) algorithm in GraphPad Prism.

Assay 4 Peripheral Selectivity

Fed male Sprague-Dawley rats (n=1/timepoint) were dosed orally with atest compound. Blood (via cardiocentesis), cerebrospinal fluid (CSF)(via cisternae magna) and brain were collected from an animal at 0.5, 1,2, 4, 8 and 24 hours post-dose. Total plasma, CSF and brainconcentrations were determined by LC-MS/MS. The pharmacokineticparameters were assessed by non-compartmental methods using WinNonlin(Version 5.3, Pharsight, Mountain View, Calif.). The unbound fraction inplasma and brain was determined by equilibrium dialysis. Free plasma andfree brain concentrations were determined by multiplying the totalconcentration in plasma or brain by its respective unbound fraction. CSFwas assumed to be unbound, due to the absence of protein. Peripheralselectivity was assessed by comparing the unbound exposures (C_(max) andAUC) in free plasma versus CSF and/or free brain.

The following test compounds were evaluated in this assay:

Free plasma AUC/ Ex. Free brain AUC 1 >70 2-7 >20 2-8 >10 3 >20Test compounds are considered to be peripherally selective if theyexhibit a ratio of free plasma concentration:free brain concentration(measured by the AUC) greater than 10.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statutes and regulations, all publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety to the same extent as if each document hadbeen individually incorporated by reference herein.

What is claimed is:
 1. A compound of formula I:

where: X and Y are —CH—, or X is —CH— and Y is —N—, or X is —N— and Y is—CH—; R¹ is selected from —C₁₋₆alkyl optionally substituted with 1 to 5groups selected from halo, —NHC(O)CH₃, and —C(O)NHCH₃; —C₃₋₆cycloalkyl;—C₁₋₂alkylene-OR; —C₁₋₂alkylene-COOR; —C₀₋₃alkylene-phenyl optionallysubstituted with —NHC(O)CH₃ or —C(O)NHCH₃; and —C₀₋₃alkylene-pyridyl;where R is selected from hydrogen and —C₁₋₃alkyl; R² is selected fromhydrogen, —C₁₋₆alkyl, and —O—C₁₋₆alkyl; R³ is selected from hydrogen,halo, and cyano; R⁴ is selected from halo; —C₁₋₆alkyl optionallysubstituted with 1 to 5 fluoro atoms; —O—C₁₋₆alkyl optionallysubstituted with 1 to 5 fluoro atoms; —C₀₋₁alkylene-phenyl;—O—C₀₋₃alkylene-phenyl; —SO₂—C₁₋₆alkyl; —C(O)NH₂; and —NO₂; and n is 1or 2; or a pharmaceutically acceptable salt thereof.
 2. The compound ofclaim 1, where R¹ is selected from methyl, ethyl, isopropyl,—(CH₂)₂NHC(O)CH₃, —(CH₂)₂OH, —(CH₂)C(O)OCH₃, phenyl, 4-phenylacetamide,and 4-pyridyl.
 3. The compound of claim 2, where R¹ is methyl.
 4. Thecompound of claim 1, where R² is hydrogen.
 5. The compound of claim 1,where R³ is selected from hydrogen and cyano.
 6. The compound of claim5, where R³ is hydrogen.
 7. The compound of claim 1, where R⁴ isselected from halo, —C₁₋₆alkyl optionally substituted with 1 to 5 fluoroatoms, —O—C₁₋₆alkyl optionally substituted with 1 to 5 fluoro atoms, and—NO₂.
 8. The compound of claim 7, where R⁴ is —CF₃.
 9. The compound ofclaim 1, where n is
 1. 10. The compound of claim 1, where X and Y are—CH—.
 11. The compound of claim 10, where R¹ is selected from —C₁₋₆alkyloptionally substituted with —NHC(O)CH₃; —C₁₋₂alkylene-OR;—C₁₋₂alkylene-COOR; phenyl optionally substituted with —NHC(O)CH₃; and-pyridyl; where R is selected from hydrogen and —C₁₋₃alkyl; R² isselected from hydrogen, —C₁₋₆alkyl, and —O—C₁₋₆alkyl; R³ is selectedfrom hydrogen and cyano; and R⁴ is selected from halo; —C₁₋₆alkyloptionally substituted with 1 to 5 fluoro atoms; —O—C₁₋₆alkyl optionallysubstituted with 1 to 5 fluoro atoms; and —NO₂.
 12. The compound ofclaim 1, where X is —CH— and Y is —N—.
 13. The compound of claim 12,where R¹ is —C₁₋₆alkyl; R² is hydrogen; R³ is hydrogen; R⁴ is selectedfrom halo and —C₁₋₆alkyl optionally substituted with 1 to 5 fluoroatoms; and n is
 1. 14. The compound of claim 1, where X is —N— and Y is—CH—.
 15. The compound of claim 14, where R¹ is —C₁₋₆alkyl; R² ishydrogen; R³ is hydrogen; R⁴ is selected from halo and —C₁₋₆alkyloptionally substituted with 1 to 5 fluoro atoms; and n is
 1. 16. Thecompound of claim 1, which has a configuration selected from:

or is enriched in a stereoisomeric form having said configuration. 17.The compound of claim 1, which has a configuration selected from:

or is enriched in a stereoisomeric form having said configuration. 18.An intermediate useful in the synthesis of the compound of claim 1,having the formula:

where P represents an amino-protecting group.
 19. A method of preparingthe compound of claim 1, the process comprising deprotecting a compoundof the formula:

to provide a compound of formula I, or a salt thereof, where P is anamino-protecting group.
 20. A pharmaceutical composition comprising thecompound of claim 1 and a pharmaceutically acceptable carrier.
 21. Thepharmaceutical composition of claim 20, further comprising a therapeuticagent selected from calcium channel blockers, endothelin receptorantagonists, PDE-5 inhibitors, prostacycline analogues, prostanoids, andcombinations thereof.